Côme Berbain

Olivier Billet

Anne Canteaut
email: anne.canteaut@inria.fr

Nicolas Courtois
email: n.courtois@ucl.ac.uk

Blandine Debraize
email: blandine.debraize@gemalto.com

Henri Gilbert
email: henri.gilbert@orange-ftgroup.com

Louis Goubin
email: louis.goubin@prism.uvsq.fr

Aline Gouget
email: aline.gouget@gemalto.com

Louis Granboulan
email: louis.granboulan@eads.net

Cédric Lauradoux
email: cedric.lauradoux@inria.fr

Marine Minier
email: marine.minier@insa-lyon.fr

Thomas Pornin
email: thomas.pornin@cryptolog.com

Hervé Sibert
email: herve.sibert@nxp.com.

Decim v2

In this paper, we present Decim v2 , a stream cipher hardwareoriented selected for the phase 3 of the ECRYPT stream cipher project eSTREAM. As required by the initial call for hardware-oriented stream cipher contribution, Decim v2 manages 80-bit secret keys and 64-bit public initialization vectors. The design of Decim v2 combines two filtering mechanisms: a nonlinear Boolean filter over a LFSR, followed by an irregular decimation mechanism called the ABSG. Since designers have been invited to demonstrate flexibility of their design by proposing variants that take 128-bit keys, we also present a 128-bit security version of Decim called Decim-128.

Introduction

Decim v2 is a hardware-oriented stream cipher selected for the phase 3 of the ECRYPT Stream Cipher Project [START_REF] Estream | Stream cipher project of the European Network of Excellence in Cryptology ECRYPT[END_REF]. Decim v2 is the tweaked version of the original submission Decim [START_REF] Berbain | Decim -A new Stream Cipher for Hardware applications[END_REF]. Decim v2 manages 80-bit secret keys and 64-bit public initialization as required by the initial eSTREAM call for contribution for the hardware-oriented profile. Decim v2 has been developed around the ABSG mechanism [START_REF] Gouget | The Bit-Search Generator[END_REF][START_REF] Gouget | Analysis of the Bit-Search Generator and sequence compression techniques[END_REF] which provides a method for irregular decimation of pseudorandom sequences. The ABSG mechanism consists of compressing the input sequence in a very simple way and it operates a highly nonlinear transformation. Being an irregular decimation, it prevents algebraic attacks and some fast correlation attacks.

The general running of Decim v2 consists first in generating a binary sequence y in a regular way from a Linear Feedback Shift Register (LFSR) which is filtered by a Boolean function. Next, the sequence y is filtered by the ABSG mechanism. Wu and Preneel found two weaknesses [START_REF] Wu | Cryptanalysis of the stream cipher decim[END_REF] in the original design of Decim that have been fixed in Decim v2 . Note that the attacks presented in [START_REF] Wu | Cryptanalysis of the stream cipher decim[END_REF] do not question the main ideas behind Decim, namely, to filter and then decimate the output of an LFSR using the ABSG mechanism. Since designers have been invited to demonstrate flexibility of their design by proposing variants that take 128-bit keys, we present a 128-bit security version of Decim called Decim-128.

The outline of the paper is as follows. In Section 2, we give an overview of Decim v2 and we detail the differences between Decim and Decim v2 . In Section 3, we provide a full description of Decim v2 . In Section 4, we explain the design rationale. In Section 5, we discuss the hardware implementation. Section 6 is dedicated to the description of Decim-128. Finally, we conclude in Section 7.

Overview of Decim v2

In accordance with the requirements given by the ECRYPT stream cipher project, Decim v2 takes as an input a 80-bit secret key and a 64-bit public initialization vector.

Keystream generation

The size of the inner state of Decim v2 is 192 bits. The keystream generation mechanism is described in Figure 1. The bits of the internal state of the LFSR are numbered from 0 to 191, and they are denoted by (x 0 , . . . , x 191 ).

The Boolean function f is a 13-variable quadratic symmetric function which is balanced. The whole filter F is a 14-variable Boolean function. The output of the function F at time t is denoted by y t . The ABSG takes as an input the sequence y = (y t ) t≥0 . The sequence output by the ABSG is denoted by z = (z t ) t≥0 . The buffer mechanism guarantees a constant throughput for the keystream; we choose a 32 bit-length buffer and the buffer outputs 1 bit for every 4 shifts by one position of the LFSR.

Key/IV setup

The Key/IV setup mechanism consists in clocking 4 × 192 = 768 times the LFSR using the nonlinear feedback described in Figure 2.

Differences between Decim and Decim v2

We do not recall in this paper the full description of the original design of Decim (see [START_REF] Berbain | Decim -A new Stream Cipher for Hardware applications[END_REF] for details). However, we briefly describe the two flaws in Decim found by Wu and Preneel [START_REF] Wu | Cryptanalysis of the stream cipher decim[END_REF], and we explain how Decim v2 fixed these two weaknesses.

The first flaw lies in the initialization stage, i.e. the computation of the initial inner state for starting the keystream generation. In Decim, at each clock of the initialization process, one of two 7-variables permutations π 1 and π 2 was applied over the internal state in order to break the linearity of the process faster. However, this mechanism could be exploited to retrieve the key. In Decim v2 , we use an initialization procedure that is both simpler and more secure than the one of Decim. In particular, the permutations are removed in Decim v2 (we refer to [START_REF] Berbain | Decim v2[END_REF] for more details).

Moreover, the number of clocks of the register during the initialization phase is increased in Decim v2 in order to ensure that the nonlinearity of the initialization stage is sufficient.

The second flaw lies in the keystream generation algorithm. More precisely, there is a flaw in Decim in the generation of the sequence y which is the output of the filter (the sequence y is next decimated by the ABSG mechanism). This flaw is due to the fact that the sequence y is directly the output of a symmetric Boolean function. Indeed, the outputs of the function associated to two input vectors which have one element in common are correlated. It was then shown in [START_REF] Gouget | Revisiting correlation-immunity in filter generators[END_REF] that the filter criterion to avoid such correlation is the quasi-immunity criterion. The choice of the filter in Decim v2 takes this design criterion into account.

Specification

In this section, we describe each component of Decim v2 .

The filtered LFSR

This section describes the filtered LFSR that generates the sequence y (the sequence y is the input of the ABSG mechanism).

The LFSR. The underlying LFSR is a maximum-length LFSR of length 192 over F 2 . It is defined by the following primitive feedback polynomial:

P (X) = X 192 + X 189 + X 188 + X 169 + X 156 + X 155 + X 132 + X 131 +X 94 + X 77 + X 46 + X 17 + X 16 + X 5 + 1 .
The sequence of the linear feedback values of the LFSR is denoted by s = (s t ) t≥0 and the recursion that corresponds to P for the LFSR is

s 192+n = s 187+n ⊕ s 176+n ⊕ s 175+n ⊕ s 146+n ⊕ s 115+n ⊕ s 98+n ⊕ s 61+n ⊕ s 60+n ⊕ s 37+n ⊕ s 36+n ⊕ s 23+n ⊕ s 4+n ⊕ s 3+n ⊕ s n .
The filter. The filter function is the 14-variable Boolean function defined by: F : F 14 2 -→ F 2 ; a 1 , . . . , a 14 → f (a 1 , . . . , a 13 ) ⊕ a 14 where f is the symmetric quadratic Boolean function defined by:

f (a 1 , . . . , a 13 ) = 1≤i<j≤13 a i a j 1≤i≤13 a i
The tap positions of the filter are:

191 -186 -178 -172 -162 -144 -111 -104 -65 -54 -45 -28 -13 -1
and the input of the ABSG at the stage t is:

y t = f (s t+191 , s t+186 , s t+178 , s t+172 , s t+162 , s t+144 , s t+111 , s t+104 , s t+65 , s t+54 , s t+45 , s t+28 , s t+13 ) ⊕ s t+1

Decimation

This part describes how the keystream sequence z is obtained from the sequence y.

The action of the ABSG on y consists in splitting y into subsequences of the form (b, b i , b), with i ≥ 0 and b ∈ {0, 1}; b denotes the complement of b in {0, 1}. For every subsequence (b, b i , b), the output bit is b for i = 0, and b otherwise. The ABSG algorithm is given in Figure 3.

Input: (y0, y1, . . . ) Set: i ← 0; j ← 0; Repeat the following steps:

1. e ← yi, zj ← yi+1;

2. i ← i + 1; 3. while (yi = e) i ← i + 1; 4. i ← i + 1;
5. output zj 6. j ← j + 1 Fig. 3. ABSG Algorithm

Buffer mechanism

The rate of the ABSG mechanism is irregular and therefore we use a buffer in order to guarantee a constant throughput. We choose a buffer of length 32 and for every 4 bits that are input into the ABSG, the buffer is supposed to output one bit exactly. With these parameters, the probability that the buffer is empty while it has to output one bit is less than 2 -89 .

If the ABSG outputs one bit when the buffer is full, then the newly computed bit is not added into the queue, i.e. it is dropped. The initial filling of the buffer is part of the initialization process detailed in 3.4.

Key/IV Setup

This subsection describes the computation of the initial inner state for starting the keystream generation. Notice that the ABSG mechanism is not used during the initialization stage.

Initial filling of the LFSR. The secret key K is a 80-bit key denoted by K = K 0 , . . . , K 79 and the initialization vector IV is a 64-bit IV denoted by IV 0 , . . . , IV 63 . The initial filling of the LFSR is done as follows.

x i =            K i 0 ≤ i ≤ 79 K i-80 ⊕ IV i-80 80 ≤ i ≤ 143 K i-80 ⊕ IV i-144 ⊕ IV i-128 ⊕ IV i-112 ⊕ IV i-96 144 ≤ i ≤ 159 IV i-160 ⊕ IV i-128 ⊕ 1 160 ≤ i ≤ 191
The number of possible initial values of the LFSR state is 2 80+64 = 2 144 .

Update of the LFSR state. The LFSR is clocked 4 × 192 = 768 times using a nonlinear feedback relation. Let y t denote the output of f at time t and let lv t denote the linear feedback value at time t > 0. Then, the value of x 191 at time t is computed using the equation:

x 191 = lv t ⊕ y t .
Notice that there is no bit of the LFSR state output during this step.

Initial filling of the buffer. After the previous step, the buffer has to be filled before starting keystream generation. In order to fill the buffer, we repeat the keystream generation process until the buffer is full. During this step, the buffer is not shifted. In particular, the buffer does not output any bit until it is completely filled. Then, the buffer is filled on average after 96 steps, and it is filled after 234 steps with probability bigger than 1 -2 -80 . Thus, if a constant duration initialization process is required, one can choose to execute 234 steps and throw away the ABSG output bits when the buffer is full.

Design rationale

In this section, we give the rationale for every component of Decim v2 .

The filtered LFSR

The LFSR. The length of the LFSR, which corresponds to the size of the internal state of the cipher, must be at least 160 in order to avoid time-memory-date trade-off attacks [START_REF] Hong | Rediscovery of Time Memory Tradeoffs[END_REF][START_REF] De Cannière | Comments on the rediscovery of Time Memory Data Tradeoffs[END_REF]. Nevertheless, we add a security margin to the LFSR length in order to deal with a reduction of the size of the potential initial state due to the initialization procedure (see Section 4.3). Therefore, we choose a 192-bit LFSR.

The choice of the primitive feedback polynomial P must be made in accordance with the following constraints. The differences between two consecutive positions of the inputs of the feedback polynomial are pairwise coprime. Furthermore, the weight of P must be large enough in order to prevent the existence of sparse multiples with low degree that could be exploited in fast correlation attacks or in distinguishing attacks. However, we do not want the weight of P to be too large, in order to reduce both the overall computational time of the cipher and its hardware size.

The feedback polynomial has been chosen carefully, i.e. it has not low Hamming weight multiples at least for the first 2 40 next degrees. However, we mention the possibility of a distinguishing attack similar to the distinguishing attack on the Self-Shrinking Generator given in [START_REF] Ekdahl | Predicting the shrinking generator with fixed connections[END_REF].

The filtering function. An important property for the filter is that the output of the filter must be uniformly distributed. Moreover, the filtering function must satisfy some other well-known cryptographic properties. Indeed, it is expected to be far from an affine function (using the Hamming distance). Moreover, the attack presented by Wu and Preneel against Decim [START_REF] Wu | Cryptanalysis of the stream cipher decim[END_REF] revealed that the filtering function must also fulfil the quasi-immunity criterion [START_REF] Gouget | Revisiting correlation-immunity in filter generators[END_REF], which is a criterion weaker than being correlation-immune of order 1.

Since Decim v2 is a hardware-oriented cipher, the Boolean filtering function must have a low-cost hardware implementation. In order to get an efficient computation of the function, the Boolean function f has been chosen to be symmetric, i.e. the value of f only depends on the Hamming weight of the input.

The symmetric Boolean functions that best fulfils the previous mentioned criteria are quadratic and have an odd number of input variables. The whole filter F of Decim v2 , constructed from a balanced 13-variable symmetric function, is balanced and correlation-immune of order 1.

The tap positions: filter and feedback polynomial. Assuming knowledge of the keystream z, an attacker will have to guess some bits of the sequence y in order to attack the function f . The knowledge of the bits of y directly yields equations in the bits of the initial state of the LFSR. Thus, the number of monomials in the bits of the initial state of the LFSR that are involved in these equations has to be maximized. Moreover, this number has to grow quickly during the first clocks of the LFSR. This implies the following two conditions:

1. each difference between two positions of bits that are input to f should appear only once; 2. some inputs of f should be taken at positions near the one of the feedback bit (which means that some inputs should be leftmost on Figure 1).

Finally, the tap positions of the inputs of the Boolean function f and the inputs of the feedback relation should be independent.

Decimation

The ABSG mechanism was first presented at the ECRYPT Workshop State of the art of stream ciphers [START_REF] Gouget | The Bit-Search Generator[END_REF] and next published in [START_REF] Gouget | Analysis of the Bit-Search Generator and sequence compression techniques[END_REF]. The ABSG is a scheme that, like the Shrinking Generator (SG) [START_REF] Coppersmith | The shrinking generator[END_REF] and the Self-Shrinking Generator (SSG) [START_REF] Meier | The self-shrinking generator[END_REF], provides a method for irregular decimation of pseudorandom sequences. The ABSG has the advantage on the one hand over the SG that it operates on a single input sequence instead of two and on the other hand over the SSG that it operates at a rate 1/3 instead of 1/4 (i.e. producing n bits of the output sequence requires on average 3n bits of the input sequence instead of 4n bits).

The best known attack on the ABSG filtering a single maximumlength LFSR [START_REF] Gouget | Analysis of the Bit-Search Generator and sequence compression techniques[END_REF][START_REF] Gouget | How to strengthen pseudo-random generators by using compression[END_REF] is based on a guess of the most favorable case. Such a guess requires ℓ output bits in order to guess 2ℓ inputs bits. The guess is correct with probability 1 2 ℓ . In order to check the correctness of his guess, the attacker should try to solve the equations in the bits of the initial state of the LFSR that arise from the bits of y he has guessed. This attack can be used in order to reconstruct 2L consecutive bits of the sequence y from L consecutive bits of the sequence z; it costs O(2 L 2 ) and requires O(L2 L 2 ) bits of z. Let Λ(y) denote the linear complexity of y. Then, the minimal length of a linear feedback shift register which generates the sequence y is Λ(y). The previous attack can be used to reconstruct the initial state of the equivalent LFSR that generates the sequence y. Then, this attack costs O(2

Λ(y)
We have checked that the linear complexity of y is the best linear complexity expected according to the choice of the Boolean function and the primitive polynomial, that is, Λ(y) = 18528.

Key/IV Setup

The components of the keystream generation are re-used for the key/IV setup; we do not introduce new components.

By using a 80-bit key and a 64-bit IV, the number of possible initial states is at most 2 144 which is the case in Decim v2 . The key schedule includes a non-linear feedback mechanism that is repeated L times, where L is the length of the register. Thus, in order to deal with the reduction of the potential internal state of the register during this phase, and considering that this non-linear feedback behaves randomly, we chose L = 192 to ensure that the final internal state is at least twice the key length, that is, 160.

The buffer mechanism

The buffer mechanism guarantees a constant throughput for the keystream. However, the buffer must have a reasonable length since the keystream generation process starts when the buffer is full.

Recall that for every α bits that are input into the ABSG, the buffer is supposed to output one bit exactly. The output rate of the ABSG is 1/3 in average. Then, the value of α is greater than 3. For α = 4 and a buffer of length 32, the probability that the buffer is empty while it has to output one bit is less than 2 -89 (the analysis of the buffer mechanism is detailed in [START_REF] Berbain | Decim -A new Stream Cipher for Hardware applications[END_REF]).

Timing measurements at the output of the keystream generator is useless since a buffer is used and the throughput is constant. However, if the attacker gets timing information from the internal keystream generator, then timing attacks apply.

Hardware implementation

There is a trade-off between the size of the hardware implementation and the throughput of the cipher. Indeed, the 32-bit length of the buffer has been chosen to ensure that the buffer is ready with probability (1 -2 -89 ) to output one bit every 4 bits entered into the ABSG.

Since each LFSR clock contributes one bit to the sequence entering the ABSG mechanism, one solution is to clock four times the LFSR before outputting one bit. The number of gates involved in an hardware implementation can be estimated as follows, based on the estimation for elementary components given in [START_REF] Batina | Energy, perfomance, area versus security trade-offs for stream ciphers[END_REF], i.e., 12 gates for a flip-flop, 2.5 gates for an XOR, 1.5 gates for an AND and 5 gates for a MUX.

-LFSR: 2339 gates corresponding to 192 flip-flops and 14 XORs.

-Filtering function: 86.5 gates corresponding to 6 Full Adders and 7

XORs (details on the hardware implementation of quadratic symmetric functions are given in [START_REF] Berbain | Decim -A new Stream Cipher for Hardware applications[END_REF]). -1-input ABSG, as described in Figure 4 Moreover, the throughput of the generator can be doubled at a low implementation cost by using a simple speed-up mechanism. This can be done with a circuit which computes two feedback bits for the LFSR, simultaneously, as described in [START_REF] Berbain | Decim -A new Stream Cipher for Hardware applications[END_REF]. This LFSR with doubled clock rate can be implemented within 192 flip-flops and 28 XORs. One additional copy of the filtering function is also required, and a 2-input ABSG mechanism must be used.

Decim-128

In this section, we describe Decim-128 which is an adaptation of the design of Decim v2 to get 128-bit security (we refer to [START_REF] Berbain | [END_REF] for more details).

Decim-128 takes as input a 128-bit secret key and a 128-bit public initialization vector. The keystream generation mechanism is similar as the one described in Figure 1 and the Key/IV setup mechanism is similar as the one described in Figure 2 except that the LFSR has length 288.

The filtered LFSR

The underlying LFSR is a maximum-length LFSR of length 288 (instead of 192) over F 2 . It is defined by the following primitive feedback polynomial:

P (X) = X 288 + X 285 + X 284 + X 247 + X 204 + X 185 + X 154 + X 125 +X 124 + X 123 + X 82 + X 35 + X 18 + X 5 + 1
The filter function is the same as in Decim v2 The sequence y produced by the filter is of maximal nonlinear complexity, namely equal to 288×289 2 = 41616.

The buffer mechanism

For Decim-128, we choose a buffer of 64 bits instead of 32. Since the buffer outputs one bit exactly for every 4 bits that are input into the ABSG, the probability that the buffer is empty while it has to output one bit is less than 2 -178 at each step.

Key/IV Setup

The secret key K is a 128-bit key denoted by K = K 0 , . . . , K 127 and the initialization vector IV is a 128-bit IV denoted by IV = IV 0 , . . . , IV 127 . The initial filling of the LFSR is done as follows.

x i = K i 0 ≤ i ≤ 127 K i-128 ⊕ IV i-128 128 ≤ i ≤ 255
We complete the register with x 256 . . . x 287 = 0x55555555. The number of possible initial values of the LFSR is 2 256 . This step slightly differs from the injection in Decim v2 . Namely, it is simpler, partly due to the fact that the key and the IV have the same size.

The update of the LFSR is done in the same way as for Decim v2 . The number of clocks performed is also 4 times the length of the LFSR, so here 4 × 288 = 1152 times. After this step, the buffer has to be filled in the same way like for Decim v2 , i.e. by performing the same steps as for keystream generation without shifting the buffer and outputting bits, until the buffer is full. Nevertheless, the buffer is filled with probability bigger than 1 -2 -128 after 432 steps, which can be used if a constant initialization time is required.

Conclusion

We have presented the stream cipher Decim v2 selected in the Phase 3 of the eSTREAM call for stream cipher profile 2, and the 128-bit security version of Decim v2 called Decim-128.

Decim v2 and Decim-128 are especially suitable for hardware applications with restricted resources such as limited storage or gate count. Design choices influence the miniaturization of the cipher system:

-the ABSG mechanism has low-cost hardware implementation, -the filtering function f only depends on the Hamming weight of its input in order to reduce the cost in hardware implementation, -the IV injection/key schedule re-uses the main components of the keystream generation mechanism.

For applications requiring higher throughputs, speed-up mechanisms can be used to accelerate Decim v2 and Decim-128 at the expense of a higher hardware complexity. Finally, the security of Decim v2 and Decim-128 mainly relies on the security of the ABSG, and there is no identified attack better than exhaustive search.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Decim v2 keystream generation

Fig. 4 .

 4 Fig. 4. Hardware implementation of the ABSG

  . The only difference between Decim v2 and Decim-128 is a different choice of tap positions:

	287, 276, 263, 244, 227, 203, 187, 159, 120, 73, 51, 39, 21, 1

Acknowledgement

This work was partially supported by the French Ministry of Research RNRT Project "X-CRYPT" and by the European Commission via the ECRYPT Network of Excellence IST-2002-507932. Note that this work was done while the 4th author was affiliated to Axalto/Gemalto (France), the 8th and the 13th authors were affiliated to France Télécom R&D/Orange Labs (France), the 9th author was affiliated to the École Normale Supérieure (France), the 11th author was affiliated to INRIA Rocquencourt (France).


Rafael Gomes

Borowski

Danielle Silva Trentin
email: daniellest@ufcspa.edu.br

Biofilms and coronaviruses reservoirs: a perspective review

Keywords: Coronaviruses, biofilm, host, bat, microbial interaction, persistent infection

Bats are a key reservoir of coronaviruses (CoVs), including the agent of the severe acute respiratory syndrome, SARS-CoV-2, responsible for the recent deadly viral pneumonia pandemic. However, understanding how bats can harbor several microorganisms without developing illnesses is still a matter under discussion. Viruses and other pathogens are often studied as stand-alone even though it is known that, in nature, they mostly live in multi-species associations called biofilms -both externally and within the host. Microorganisms in biofilms are enclosed by an extracellular matrix that confers protection and improves survival. Previous studies have shown that viruses can secondarily colonize preexisting biofilms, and viral biofilms have also been already described. In this review, we raise the perspective that CoVs can persistently infect bats due to occurrence in biofilm structures. This phenomenon potentially provides an optimal environment for non-pathogenic and well-adapted viruses to interact with the host, as well as for viral recombination. Biofilms can also enhance virion viability in extracellular environments, such as in fomites and aquatic sediments, allowing viral persistence and dissemination. Moreover, understanding CoVs biofilm lifestyle in reservoirs might contribute to explain several burning questions that remain unanswered including persistence and transmissibility by highly pathogenic emerging CoVs.

Coronaviruses

Human activities, including urbanization and, increase in global commerce, have led to drastic ecological changes [START_REF] Schlesinger | Global change ecology[END_REF][START_REF] Palmer | Ecology. Ecology for a crowded planet[END_REF]. This phenomenon, allied to social and cultural behaviors may affect the balance between health and disease [START_REF] Bavel | Using social and behavioural science to support COVID-19 pandemic response[END_REF]. As consequence, we have seen the successful introduction of animal pathogens to the human population, such as the zoonotic transmission of non-primate viruses [START_REF] Bastone | Potential of zoonotic transmission of non-primate foamy viruses to humans[END_REF]. Coronaviruses (CoVs) have been classified as zoonotic pathogens that infect the respiratory, gastrointestinal, hepatic, and central nervous systems of unrelated hosts. A historical and evolutionary overview of CoVs (1930CoVs ( -2020) ) transmission to hosts are summarized in Box 1 and Figure 1.

CoVs are enveloped viruses that belong to the Coronaviridae family [START_REF] Chen | Emerging coronaviruses: Genome structure, replication, and pathogenesis[END_REF]. The disease-related CoVs in humans including the severe acute respiratory syndrome coronavirus (SARS-CoV), the new SARS-CoV-2 and the Middle East respiratory syndrome coronavirus (MERS-CoV) are classified as Betacoronavirus. They are positive-strand RNA viruses owing large genomes (30-32 kb) made of proteins such as the spike and transmembrane glycoproteins, the envelope protein, the nucleocapsid and, some of them, the haemagglutinin-esterase [START_REF] Perlman | Coronaviruses post-SARS: update on replication and pathogenesis[END_REF][START_REF] Neuman | Supramolecular Architecture of the Coronavirus Particle[END_REF][START_REF] Hu | Characteristics of SARS-CoV-2 and COVID-19[END_REF].

Box 1 -Chronological overview of CoVs

Probably first observed in the '30s, it was in 1968 that Nature published the description of a cluster of viruses found in humans and animals with the appearance of the "solar corona." These viruses contained a characteristic "fringe", supported by electron microscopy. The International Committee for the Nomenclature of Viruses (ICNV) named them coronaviruses (5, 6). In the '70s the attention to CoVs was focused on the veterinary field, especially for calves diseases that were causing losses in animal production and economy [START_REF] Mcclurkin | Probable role of viruses in calfhood diseases[END_REF]. It was in the 80's we have acquired more scientific information about these viruses according to clinical significance. A book was published summarizing the status quo of coronavirus research [START_REF] Lai | Molecular biology of coronavirus 1986[END_REF]. The next decade was highlighted by advances in molecular and structural biology knowledge and insights about human infections began to appear [START_REF] Mostl | Coronaviridae, pathogenetic and clinical aspects: an update[END_REF]; demonstrating an expansion of the spectrum of animals that could be infected by them. Furthermore, the community-acquired human CoVs strains 229E and OC43 were characterized. Since 2003, several studies have been present in the literature. This occurred as a reflection of the Asia CoV epidemic (SARS) in 2002. In 2012, a novel CoV was reported on the Program for Monitoring Emerging Diseases (ProMEDmail), named later as the Middle East respiratory syndrome-coronavirus (MERS-CoV) [START_REF]Middle East Respiratory Syndrome-coronavirus infection: an overview[END_REF][START_REF] Al-Tawfiq | Emerging respiratory and novel coronavirus 2012 infections and mass gatherings[END_REF]. MERS was first identified in humans in Saudi Arabia [START_REF] Bermingham | Severe respiratory illness caused by a novel coronavirus, in a patient transferred to the United Kingdom from the Middle East, September 2012[END_REF]. Sporadic outbreaks have been reported until now in African and European countries and the United States of America, but exclusively in individuals traveling back from the Middle East [START_REF] Who | Middle East Respiratory Syndrome Coronavirus[END_REF]. Recently, 2019 raised the "tip of the iceberg" that would become the 2020 declared pandemic by WHO. A respiratory disease of unknown etiology was firstly detected in Wuhan, Hubei Province, China, and started to circulate the world [START_REF] Who | Novel coronavirus 2019[END_REF]. Chinese scientists identified the etiological agent as a new coronavirus, the 2019-nCoV [START_REF] Zhou | A Novel Bat Coronavirus Closely Related to SARS-CoV-2 Contains Natural Insertions at the S1/S2 Cleavage Site of the Spike Protein[END_REF] which was renamed SARS-CoV-2. Subsequently, the disease name was recommended as COVID-19 by the WHO. Clinical signs, similar to SARS and MERS, such as fever, cough and difficulties in breathing, with evolution to severe pneumonia and organ failures, were verified in the most drastic cases [START_REF] Huang | Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China[END_REF]. Only in 2020, more than 37,204 articles were published on this subject (15 th October 2020 from PubMed) and the emergence of human pathogenic CoVs attend a constant discussion.

Although there are different hypotheses about the origin of the virus [START_REF] Sallard | Tracing the origins of SARS-COV-2 in coronavirus phylogenies: a review[END_REF], the close phylogenetic relationship with bats CoVs provides strong evidence that SARS-CoV-2 may be originated from these animals and that they are the reservoir host of this virus. It has been proposed that the SARS-CoV-2 was introduced into a small cluster of humans from infected animals and, from there, the virus acquired the capacity for human-to-human transmission. Pangolins could be one of the intermediate amplifying hosts for the virus, which is surprising since they are solitary animals with relatively small population sizes, reflecting their endangered status [START_REF] Heinrich | THE GLOBAL TRAFFICKING OF PANGOLINS: A comprehensive summary of seizures and trafficking routes from 2010-2015[END_REF]. However, human exposition to these animals exists, since they are recognized as a meat delicacy sold in the parallel market and their scales are utilized in traditional Chinese medicine.

Regarding CoVs, the source of the viruses, their persistence, reservoirs and hosts, and the transmission to humans are topics under continuous discussion in the scientific community, and will also be addressed by this review-perspective study. There are advanced studies that establish the role of biofilms in microbial persistence, resistance and latent infections but to date, none of them bring up the potential involvement of biofilms in CoVs reservoirs (bats and environment). Overall, bats have been shown to possess an evident role in these processes, especially as the natural reservoir of diverse viruses. However, several attractive questions remain to be answered, such as (i) How do CoVs and other viruses persistently infect bats? (ii) How do they evade the immune system of bats? (iii) How are they not pathogenic for bats while leading to lethal human disease? (iv) How do they undergo several recombination events, favoring the emergence of distinct genetic variants from bats? And (v) How do they remain infectious in the environment, at least for hours or days, after being expelled in secretions by a contaminated host? This perspective review shed new light on complex host-virus-environmental interactions.

Coronaviruses and hosts

For many years the four community-acquired CoVs (i.e. HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63) circulate in the human population triggering common cold in immunocompetent subjects, and, likely, these viruses do not depend on animal reservoir [START_REF] Ye | Zoonotic origins of human coronaviruses[END_REF]. In contrast, highly pathogenic CoVs (SARS-CoV, MERS-CoV, and SARS-CoV-2) are maintained and propagated in their zoonotic reservoirs and eventually spillover to susceptible human targets, possibly via one or more intermediate and amplifying hosts (summarized in Boxes 2-4).

Box 2 -SARS-CoV

The first SARS-CoV patients reported exposure to animal trade in wet markets or in restaurants where live animals were kept in Guangdong Province -China. Then, the SARS-CoV-like virus was isolated from Himalayan palm civets (Paguma larvata) presenting 99.8% of genomic identity with that from humans [START_REF] Guan | Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China[END_REF][START_REF] Song | Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human[END_REF]. Although 80% of other animals from nearby markets possessed anti-SARS-CoV antibodies [START_REF] Tu | Antibodies to SARS coronavirus in civets[END_REF], the massive culling of market civets played a major role in the efficient control of SARS. This indicates that palm civets might serve as the intermediate amplifying host but not as the natural reservoir for SARS-CoV. Multiple species of mammals might also serve as intermediate and dead-end hosts for SARS-CoV [START_REF] Ye | Zoonotic origins of human coronaviruses[END_REF].

Since 2005 a large number of SARS-related CoVs (SARSr-CoVs) have been detected in horseshoe bat species (Rhinolophus spp.) in China [START_REF] Fan | Bat Coronaviruses in China[END_REF]. A 5-year surveillance study reported distinct sequences of SARSr-CoVs in different genes from multiple species of horseshoe bats. This suggests that bat viruses are considered unlikely to represent the direct progenitor of the human virus. Importantly these bat SARSr-CoV strains present different S proteins but all of them might interact with the cell entry receptor in humans, the angiotensinconverting enzyme II -ACE2 [START_REF] Hu | Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus[END_REF]. Likewise, another study also showed that SARSr-CoVs from bats faecal samples use ACE2 receptor (from humans, civets, and horseshoe bats) for cell entry, indicating a viral broad species tropism [START_REF] Ge | Metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in China[END_REF].

These findings likely explain that the direct ancestor of SARS-CoVs has arisen from sequential recombination events within bats before spillovers to an intermediate host, such as farmed civets or another mammal, which transmitted the virus to other civets by faecal-oral transmission. So, it is likely that when virusinfected civets were transported to Guangdong markets, the virus spread to market civets and acquired further mutations before spillover to humans [START_REF] Cui | Origin and evolution of pathogenic coronaviruses[END_REF].

Box 3 -MERS-CoV

Bats also have been proposed to harbor the progenitor viruses of MERS-CoV, although human infection has been linked to contact with dromedary camels (Camelus dromedarius) or other infected-humans [START_REF] Chan | Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease[END_REF]. The Middle East is a region that possesses religious and cultural practices along with a reliance on camels for food, medicine, and travel. As reviewed by Cui and collaborators (2019), specific antibodies were highly prevalent in these animals from that region and their MERS-CoV strains were almost identical to those from humans (> 99% identity). Furthermore, infectious MERS-CoVs have been isolated from camels [START_REF] Raj | Isolation of MERS coronavirus from a dromedary camel, Qatar, 2014[END_REF] and the respiratory route, flesh, and discharges, such as faeces and milk [START_REF] Ye | Zoonotic origins of human coronaviruses[END_REF] are recognized as possible transmission ways to humans. Nevertheless, several patients did not have an epidemiological link to infected camels or humans [START_REF] Chan | Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease[END_REF]. In this sense, several animal species including livestock harbor the human MERS-CoV dipeptidyl peptidase 4 (DPP4) receptor, however only alpaca (Vicugna pacos) and goat (Capra hircus) cells were showed to be permissive for MERS-CoV replication in comparison to sheep, cattle, and rodent cells [START_REF] Eckerle | Replicative Capacity of MERS Coronavirus in Livestock Cell Lines[END_REF].

MERS-related CoVs (MERSr-CoVs) were found in bat species from two bat families [START_REF] Fan | Bat Coronaviruses in China[END_REF][START_REF] Cui | Origin and evolution of pathogenic coronaviruses[END_REF] however, none of these viruses is recognized as a direct progenitor of MERS-CoV since their S proteins differ substantially. Overall, all the MERSr-CoVs isolated from bats support the hypothesis that they are originated from these mammals followed by numerous recombination events that implied the exchange of genetic elements between different viral ancestors, including camels and bats [START_REF] Cui | Origin and evolution of pathogenic coronaviruses[END_REF]. Bat viruses have been detected in urine, faeces, saliva, and soiled fruit, indicating the potential for virus transmission via bats excreta in the wild. Emerging bats feed on palms or other crops and those seeds may carry bat faeces, possibly possessing MERS-CoVs. When seeds drop to the ground they might be eaten by camels [START_REF] Fakhoury | Re-emerging Middle East respiratory syndrome coronavirus: The hibernating bat hypothesis[END_REF].

Pervasive evidence has shown that many of the human and animal CoVs have a bat origin. They are transmitted to humans via intermediate hosts although the crossspecies pathways are still unknown [START_REF] Fan | Bat Coronaviruses in China[END_REF][START_REF] Banerjee | Bats and Coronaviruses[END_REF]. Bats have been recognized as special reservoirs for viral pathogens since the ancient phylogenetic relationship between viruses from bats and a variety of other human viral pathogens has been confirmed [START_REF] Drexler | Amplification of emerging viruses in a bat colony[END_REF][START_REF] Quan | Bats are a major natural reservoir for hepaciviruses and pegiviruses[END_REF][START_REF] Ciminski | Bats reveal the true power of influenza A virus adaptability[END_REF].

Mammalian host-virus relationships demonstrated that bats harbor a significantly higher proportion of zoonotic viruses than other mammalian orders. Bats account for more than 20% of all classified mammal species worldwide and they possess wide

Box 4 -SARS-CoV-2

Viral full-length genome sequences obtained from five patients at the early stage of the outbreak were almost identical among them and shared only 79.6% sequence identity to SARS-CoV. These CoV genomes presented 96.2% sequence identity to a bat CoV (BatCoV RaTG13) detected in Rhinolophus affinis bats from China, leading to the identification of the SARS-CoV-2 [START_REF] Zhou | A pneumonia outbreak associated with a new coronavirus of probable bat origin[END_REF]. SARS-CoV2 also uses ACE2 as a cell entry receptor. It is capable of using the ACE2 proteins from humans, Chinese horseshoe bats, civets, and pigs and it does not bind to DPP4 receptors, used by MERS-CoV 2 [START_REF] Zhou | A pneumonia outbreak associated with a new coronavirus of probable bat origin[END_REF]. However, BatCoV RaTG13 might be unconsidered the immediate ancestor for SARS-CoV-2 because the genetic sequence encoding for ACE2 shares only 89% of identity with that of SARS-CoV-2. Recently, another batderived CoV denoted RmYN02 from Rhinolophus malayanus was identified. This virus shares 93.3% of identity with SARS-CoV-2 for the complete genome and 97.2% for the 1ab gene, which is the closest relative of SARS-CoV-2 reported to date. In contrast, RmYN02 might not bind to the ACE2 receptor [START_REF] Zhou | A Novel Bat Coronavirus Closely Related to SARS-CoV-2 Contains Natural Insertions at the S1/S2 Cleavage Site of the Spike Protein[END_REF]. Presumably, the intermediate animal hosts of SARS-CoV-2 should be among the wildlife species sold at the Huanan Seafood Wholesale Market where many of the initial cases of COVID-19 were associated, indicating an animal-to-human transmission event.

Therefore, the origins of this virus have not been conclusively ( 23) and possibly has more than one source of infection. SARSr-CoVs were also identified from 25 Malayan pangolins (Manis javanica) collected from a wildlife rescue center [START_REF] Xiao | Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins[END_REF][START_REF] Wahba | An Extensive Meta-Metagenomic Search Identifies SARS-CoV-2-Homologous Sequences in Pangolin Lung Viromes[END_REF]. Because of the sequence divergences over the whole genome, phylogenetic analyses did not support that the SARS-CoV-2 arose directly from the pangolin-CoV [START_REF] Liu | Overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses SARS-CoV, MERS-CoV, and 2019-nCoV[END_REF], but evidence suggests it might have originated from the recombination of a pangolin-CoV-like virus with a Bat-CoV-RaTG13-like virus. geographical mobility and distribution, and extensive species diversity [START_REF] Fan | Bat Coronaviruses in China[END_REF]. They are the only mammals to have achieved true self-powered flight. This enables them to have a longer range of migration as well as excrements spread and intimate interactions with humans and livestock when compared to land ones [START_REF] Fan | Bat Coronaviruses in China[END_REF][START_REF] Teeling | A molecular phylogeny for bats illuminates biogeography and the fossil record[END_REF]. The migratory ability of bats likely has particular relevance in the context of disease transmission. Furthermore, humans might share some ecological niches with bats through butchering or coal mining; they are consumed as game meat and their excrements, such as faeces, are often used in traditional Chinese medicine [START_REF] Wassenaar | _nCoV/SARS-CoV-2: rapid classification of betacoronaviruses and identification of Traditional Chinese Medicine as potential origin of zoonotic coronaviruses[END_REF]. The Chinese food culture beliefs that live slaughtered animals are more nutritious, potentially enhancing the risks of viral transmission [START_REF] Fan | Bat Coronaviruses in China[END_REF].

Biofilm and viruses

It is established that the majority of microorganisms on earth live in biofilms [START_REF] Flemming | Bacteria and archaea on Earth and their abundance in biofilms[END_REF][START_REF] Kernien | The Interface between Fungal Biofilms and Innate Immunity[END_REF]. Viruses play an extensive ecological role and have been reported to exist in diverse microbial communities such as biofilms. They are involved in several dynamics such as microbial diversity and biogeochemical cycles due to their prevalence and variation across diverse ecosystems [START_REF] Mateus | Bridging the Gap between Knowing and Modeling Viruses in Marine Systems-An Upcoming Frontier[END_REF]. Therefore, it is essential to comprehend how the viruses effectively persist in different environments because this process can assist in the understanding of their transmissibility and cross-infectivity among different hosts. At this moment, there is limited knowledge about the relationship between viruses and biofilms, which usually addresses enteric viruses.

Biofilm is an ecological cluster of microorganisms, surface-attached or floating, with the evolutionary purpose given the protection, nutrition, or strengthening survival.

In biofilms, the microorganisms are surrounded by a complex assembled extracellular matrix. This results in heterogeneous biomolecular and biochemical arrangements that hinder the entry of exogenous components while facilitates the exchange of genetic elements among microbial cells [START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Olsen | Biofilm-specific antibiotic tolerance and resistance[END_REF]. The consequences of pathogenic biofilm formation are calamitous for human health, water systems, food, and other industries and consequently for the economy [START_REF] Flemming | Biofouling and me: My Stockholm syndrome with biofilms[END_REF].

Reports in the literature about viruses in biofilms are recent, dating from the late 90s. Specifically, enteroviruses have been detected from biofilms. In 1997 a study with the nonenveloped Poliovirus-1, using an artificial water flow distribution system, brought up the discussion about virus persistence and biofilm. They showed a greater amount of viruses recovered from biofilms. Notably, those viruses seemed to be protected against the disinfectant chlorine in the opposite of those in water phase counterparts [START_REF] Quignon | Poliovirus-1 inactivation and interaction with biofilm: a pilot-scale study[END_REF]. Enterovirus and Norovirus, nonenveloped viruses, were also identified into biofilms from drinking water and wastewater samples and infectious viruses could be released from these biofilms. Besides, virions from wastewater were characterized as being more persistent when associated with biofilm, remaining infectious for up to 30 days. These results warned for the risk of virus-containing biofilms that contribute to temporal dispersion of viral-pollution in water environments [START_REF] Skraber | Occurrence and persistence of enteroviruses, noroviruses and F-specific RNA phages in natural wastewater biofilms[END_REF][START_REF] Storey | Persistence of two model enteric viruses (B40-8 and MS-2 bacteriophages) in water distribution pipe biofilms[END_REF][START_REF] Helmi | Interactions of Cryptosporidium parvum, Giardia lamblia, vaccinal poliovirus type 1, and bacteriophages phiX174 and MS2 with a drinking water biofilm and a wastewater biofilm[END_REF]. Additionally, biofilm-associated enteroviruses developed attached to pipeline walls are flow pressure and disinfectant resistant and may represent 95% of the overall potential contaminant biomass in direct contact with the water [START_REF] Flemming | Contamination potential of biofilms in water distribution systems[END_REF]. In this sense, the entitled article "Enteric virions and microbial biofilmsa secondary source of public health concern?" enquire about the presence of pathogenic virions within biofilms from distribution pipes and their neglected monitoring [START_REF] Storey | Enteric virions and microbial biofilms--a secondary source of public health concern?[END_REF]. It is important to mention the detection of SARS-CoV-2 RNA in wastewater [START_REF] Ahmed | First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community[END_REF]. Although the isolation of the infectious virus from faeces was described, no cases of faecal-oral transmission have been reported [START_REF] Wang | Detection of SARS-CoV-2 in Different Types of Clinical Specimens[END_REF].

Viruses as well as other micro-organisms may come into contact with preexisting biofilms and accidentally adhere and make part of them, thus constituting secondary colonizers. A study of 2002 supports that biofilms may encompass a set of nonenveloped enteric viruses, including Calicivirus, Rotavirus, Astrovirus, and hepatitis A virus, among other microorganisms such as Gram-negative bacteria and filamentous fungi [START_REF] Flemming | Contamination potential of biofilms in water distribution systems[END_REF]. Some authors indicate that the human oral cavity may be an active site of infection and reservoir for SARS-CoV-2 assuming its interaction with the host oral microbiota [START_REF] Xiang | Potential implications of SARS-CoV-2 oral infection in the host microbiota[END_REF], which is mostly in the form of biofilm. Importantly, both virions and virus-infected eukaryotic cells embedded in biofilms have been reported to retain their infectivity. A study investigated the enveloped virus Herpes simplex virus type 1 (HSV-1) and the nonenveloped virus Coxsackievirus type B5 (CVB5) within the fungal Candida albicans biofilms [START_REF] Mazaheritehrani | Human pathogenic viruses are retained in and released by Candida albicans biofilm in vitro[END_REF]. The authors recovered high virus titers from in vitro cultures of the virus-exposed biofilms after extensive washes, indicating that virions and viruses are deeply dispersed and protected into the fungi-produced biofilm matrix.

Still, the authors related that biofilm reduced virus sensitivity to chemical inactivation.

They discussed some mechanisms that may lead to virions inclusion into the fungal biofilm; for example, by sorption sites from the extracellular matrix, by the natural colloidal characteristic of viruses, or by biomolecules present on biological fluids. Since these viruses encompassed on biofilm kept their viability and infectivity, it indicates that biofilm lifestyle does not limit viral dissemination [START_REF] Mazaheritehrani | Human pathogenic viruses are retained in and released by Candida albicans biofilm in vitro[END_REF] and even can improve it.

Therefore, viruses may be stored in biofilms regarding temporary or long-term reservoirs in the environment [START_REF] Wingender | Biofilms in drinking water and their role as reservoir for pathogens[END_REF]. As pointed by Vasickova and collaborators (2010), the potential of viral spreading via contaminated surfaces depends particularly on the ability of the virus to maintain infectivity while it is in the environment, and they indicated some benefits for viruses within biofilms, such as protection against desiccation and antimicrobial agents [START_REF] Vasickova | Issues Concerning Survival of Viruses on Surfaces[END_REF].

A pioneering study conducted by French scientists using the enveloped human Tcell leukemia virus type 1 (HTLV-1) showed that the biofilm-like extracellular viral assemblies mediate the HTLV-1 cell-to-cell transmission [START_REF] Pais-Correia | Biofilm-like extracellular viral assemblies mediate HTLV-1 cell-to-cell transmission at virological synapses[END_REF]. Notably, the extracellular state represents a crucial step for viral dissemination since intracellular formed virions have to spread the surface of other target cells to re-start viral replication. At this moment they are exposed to physicochemical and host immune system challenges. To overcome it the authors proposed that the observed "extracellular viral assemblies" would correspond to a "viral biofilm." Viral biofilms were then defined as a protective form of "virion community embedded in a matrix of highly glycosylated proteins" with enhanced persistence, infectious capacity, and dissemination [START_REF] Thoulouze | Can viruses form biofilms?[END_REF]. They also detail the similarities of these extracellular viral assemblies and the composition, organization, and dissemination to bacterial biofilms.

To conclude, the authors pointed that it is likely a similar mechanism can be used also by other enveloped viruses, and CoVs possess enveloped capsid.

A growing number of studies reports that enveloped and non-enveloped viruses can spread in groups in so-called collective infectious units [START_REF] Sanjuán | Collective Infectious Units in Viruses[END_REF][START_REF] Sanjuán | Why viruses sometimes disperse in groups?[END_REF][START_REF] Andreu-Moreno | Collective Viral Spread Mediated by Virion Aggregates Promotes the Evolution of Defective Interfering Particles[END_REF]. The vehicles mediating collective spread vary widely and include lipid vesicles, protein matrices, diverse forms of aggregation, and binding to the surface of host or non-host cells [START_REF] Sanjuán | Why viruses sometimes disperse in groups?[END_REF].

In accordance, Tozzi and co-workers (2020) described the collective arrangements of multiple SARS-CoV-2 particles based on microscopic image analysis of viruses emerging from the surface of cultured cells. They suggest that when the virions exit the host cells, single particles tend at first to aggregate in spherical assemblies, then to develop interconnected network-like branches and nodes and this time called collective clustering dynamics [START_REF] Tozzi | Collective Clustering Dynamics of SARS-COV-2 Particles[END_REF]. However, further investigations are needed to characterize the presence of the matrix for SARS-CoV-2, such as in the study developed by Thoulouze and colleagues (2011) for HTLV-1. In this sense, the ability of Betacoronaviruses to bind carbohydrates has been mapped due to their high saccharides or carbohydrate domains and glycosylation patterns [START_REF] Belouzard | Mechanisms of coronavirus cell entry mediated by the viral spike protein[END_REF][START_REF] Balzarini | Carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses?[END_REF][START_REF] Balzarini | Targeting the glycans of glycoproteins: a novel paradigm for antiviral therapy[END_REF]. Likewise, we speculate that the affinity of CoVs virions to polysaccharides presented on the matrix of biofilms probably would play a role in secondary colonization. Moreover, would this high saccharide profile be involved in the formation of viral biofilms? This set of evidence allows us to propose that CoVs also could be present in biofilm patterns, either as viral biofilm or as a secondary colonizer of a microbial biofilm (Figure 2). Moreover, previous studies addressed in this review indicate that the (i) viral particles comprising a viral biofilm are embedded in an extracellular matrix from the host cell microenvironment, eventually, induced by the viral infection; otherwise, (ii) viral particles that are inside a microbial biofilm are surrounded by an extracellular matrix produced and externalized by bacterial and/or fungal cells.

Since viruses are strict intracellular parasites, we hypothesize that they will be unable to proliferate in biofilms, but they would persist infectious in a reservoir host due to the advantages conferred by the biofilm structure. Furthermore, the maintenance of viral particles inside biofilms facilitates the co-infection process due to physical proximity. When viruses spread collectively, they should locally increase the number of viral genomes that initiate a cell infection, which may contribute to increasing infectivity and may favor co-infections [START_REF] Sanjuán | Why viruses sometimes disperse in groups?[END_REF][START_REF] Andreu-Moreno | Collective Viral Spread Mediated by Virion Aggregates Promotes the Evolution of Defective Interfering Particles[END_REF]. To co-infect the same cell is beneficial for microorganisms since it may provide diverse genetic materials, favoring mutations and recombinations, biological diversity, and drug resistance (72) (Figure 2, step 3b).

Subsequently, we expect that CoVs-containing biofilms may also act as a viral environmental reservoir, including under fomites, and contribute to viral persistence and dissemination [START_REF] Wingender | Biofilms in drinking water and their role as reservoir for pathogens[END_REF].

Importantly, these circumstances may increase viral infectivity and favor coinfections [START_REF] Bakaletz | Viral-bacterial co-infections in the respiratory tract[END_REF]. A retrospective study from Jiangsu Province showed that 242 (94.2 % of 257) SARS-CoV-2 positive patients were co-infected with one or more pathogens.

Bacterial co-infections were dominant and Streptococcus pneumoniae, Klebsiella pneumoniae, and Haemophilus influenzae were the most common species. Additionally, viral, fungal, and bacterial-fungal co-infections were present in the highest severe SARS-CoV-2 cases [START_REF] Zhu | Co-infection with respiratory pathogens among COVID-2019 cases[END_REF]. In accordance, another study from Renmin Hospital of Wuhan University including 354 SARS-CoV-2 positive patients concluded that 24.4% of critical cases were co-infected with other respiratory pathogens. These conclusions suggest that co-infections are associated with the severity of SARS-CoV-2 (75).

Can microbial biofilms favor viral reservoirs in bats?

Bats harbor a rich pool of virus species, increasing the probability of cell coinfections. It enables the interspecies exchange of genetic fragments and the emergence of new viruses. Longevity, densely packed colonies, close social interaction, and strong ability to fly are all favorable conditions for bats to be an ideal virus spreader. It has been proposed that bats coexist with pathogens due to adaptations inadvertently acquired during the evolution of flight [START_REF] Zhang | Comparative analysis of bat genomes provides insight into the evolution of flight and immunity[END_REF]. Moreover, multiple pieces of evidence support an evolutionary origin of all human CoVs from bats.

In 2016, Plowright and colleagues described three hypotheses that dominate the current research on emerging bat infections. They pointed that pulses of viral excretion could be generated by episodic shedding from persistently infected bats through a combination of physiological and ecological factors. In this scenario, the viral acute infection is resolved without clearance of viruses which allows pulses of transmission. This is triggered by viral reactivation and the stable carriage of the pathogen in space and time, even if viral shedding is episodic. For example, enveloped Lyssaviruses and Flaviviruses persistently have infected bats and shed viruses for extensive periods without evidence of disease [START_REF] Sulkin | Virus infections in bats[END_REF]. In 2017, Jeong and co-workers demonstrated that persistent infections support the maintenance of CoVs in a bat population. It was assumed that temporally synchronized stressors such as pregnancy or poor nutrition may weaken the immune system of bats facilitating the viral reactivation, the pulses of viral shedding, and the spillover of bat-borne viruses [START_REF] Plowright | Transmission or Within-Host Dynamics Driving Pulses of Zoonotic Viruses in Reservoir-Host Populations[END_REF][START_REF] Jeong | Persistent infections support maintenance of a coronavirus in a population of Australian bats (Myotis macropus)[END_REF]. Several hurdles including the necessity of biosecurity level 4 conditions, the difficulty in reproducing natural infections in bat through experimental infections, and in isolating infectious virus from bats have constrained the understanding of emerging bat viruses [START_REF] Dominguez | Detection of group 1 coronaviruses in bats in North America[END_REF]. However, the mechanisms including physiological and ecological factors which drive infectious disease dynamics in bats such as viral persistence, clearance, and transmission without apparent disease remain poorly characterized.

Based on this set of evidence and the features regarding biofilms, we raise the perspective that some viruses, such as CoVs, can persistently infect reservoir hosts, including bats, due to biofilm lifestyle. This assumption may contribute to response some important questions and is supported by the following observations and discussions:

1 -In persistent infections, a virus is not cleared from the host, but remains associated with specific cells for long periods [START_REF] Jeong | Persistent infections support maintenance of a coronavirus in a population of Australian bats (Myotis macropus)[END_REF].

Biofilms are recognized as an important way to maintain microbial cells or particles in metabolic latency while they are protected by an external matrix. In this sense, as pointed by Thoulouze and Alcover (2011) viral biofilms could represent "viral communities" with enhanced infectious capacity and improved spread compared with "free" viral particles. This might constitute a key reservoir for chronic infections.

Additionally, as discussed by Dawley and Gibson (2019) specific associations of viruses with bacteria or other micro-organisms are likely to allow the easier entry of virions into the biofilm resulting in a reservoir of viruses. These virions are as difficult to remove and inactivate as their bacterial counterparts, turning infections persistent for a long time [START_REF] Dawley | Virus-Bacteria Interactions: Implications for Prevention and Control of Human Enteric Viruses from Environment to Host[END_REF].

-If bats do not clear infections and viruses may persist within bat hosts, it is assumed that bat hosts may tolerate viruses. In this case, a tempered inflammatory response would minimize immunopathology and viral replication may fluctuate as a function of the immunological competence of the host (78).

It has been reported that bats host intracellular microorganisms such as protozoa (Plasmodium spp.), bacteria (Bartonella spp.), and fungi (Histoplasma capsulatum), without ostensible disease symptoms. By contrast, pathogens that predominantly occupy the extracellular space present considerable challenges for bat immune systems (82).

The immune system of bats appears to be different in some features from those of other mammals and avoid an over-induction of inflammation [START_REF] Subudhi | Immune System Modulation and Viral Persistence in Bats: Understanding Viral Spillover[END_REF]. In this point, is important to mention the inflammatory process also may be inhibited by biofilms, since the surrounding matrix hinders the recognition of microorganisms by the immune system.

Additionally, once viruses are encompassed on biofilms, they will be unable to replicate. These biofilm characteristics may also explain the controlled rate of viruses, the persistent infection, the lower activation of the immune system, and the absence of active disease in bats.

-Genomic recombination events have been shown for several viruses, including

CoVs, that are harbored by bats [START_REF] Ye | Zoonotic origins of human coronaviruses[END_REF]. Compared to other RNA viruses, the expanded genome size of CoVs facilitates the acquisition of genes encoding accessory proteins that are beneficial for CoVs adaptation to a specific host and as a result, genome changes caused by recombination, gene interchange, and insertion or deletion are common among CoVs [START_REF] Fan | Bat Coronaviruses in China[END_REF]. This genetic plasticity is important to inter-species persistence-survival mechanisms [START_REF] Menachery | Jumping species-a mechanism for coronavirus persistence and survival[END_REF].

Biofilms may contribute to viral recombination events especially for supporting an environment in which distinct and infectious viruses are physically close and in the presence of extracellular enzymes and genetic fragments. These viruses would be released from the biofilm and would co-infect the same host cell leading to gene exchanges and recombination. Therefore, the facilitated cell co-infection by diverse viruses allows the evolution and emergence of new viral particles that can undergo spillover events to other animals.

In this sense, it was demonstrated that secondary infection with the white-nose syndrome fungus (Pseudogymnoascus destructans) increased CoV replication in bats [START_REF] Davy | White-nose syndrome is associated with increased replication of a naturally persisting coronaviruses in bats[END_REF]. This study opens up a new perspective about infection dynamics and microbial interactions. It demonstrates that a complex regulation possibly modulates bat responses to virus replication through their translocation from the biofilm (related to persistent infections) to virion free-living state (related to active infection) and vice-versa.

-It has been reported the difficulty of isolating infectious viruses from bats,

which may yield false-negative samples. Moreover, it has been suggested that persistent pathogens within their hosts may be sequestered in tissues while viremia or shedding in excreta may be periodic, justifying the false negatives status [START_REF] Plowright | Transmission or Within-Host Dynamics Driving Pulses of Zoonotic Viruses in Reservoir-Host Populations[END_REF][START_REF] Leroy | Fruit bats as reservoirs of Ebola virus[END_REF].

It is important to point out that this technical obstacle also may be imposed if viruses are enclosed within biofilm structures.

Do biofilm mediate CoVs extracellular viability?

Viruses dissemination closely depends on hosts and environmental interactions. SARS-CoV and -CoV-2 human transmission can occur with both direct (air-borne and droplets) and indirect contact (contaminated surfaces) between susceptible and infected individuals [START_REF] Boone | Significance of fomites in the spread of respiratory and enteric viral disease[END_REF][START_REF] Spicknall | Informing optimal environmental influenza interventions: how the host, agent, and environment alter dominant routes of transmission[END_REF]. For this, viruses must be shed into the environment and contaminate biotic (mucosa) or abiotic (non-living structures like fomites) surfaces.

Notably, they must be capable to remain infectious on that for at least a time (Figure 3).

In 2017, a review addressed the different types of viruses' collective spread and pointed the advantages of viral dissemination through "complex multi-virion structures", such as viral biofilms and viruses as secondary colonizers, in comparison to free individual virions [START_REF] Sanjuán | Collective Infectious Units in Viruses[END_REF][START_REF] Sanjuán | Why viruses sometimes disperse in groups?[END_REF]. In this context, we point out two leading biofilm features that could directly or indirectly afford and maintain virus viability through some intrinsic abilities:

1 -Biofilm may act as viral storage where virions remain infectious within the hosts (wild, livestock or domestic animals and humans) and on the environment (food, water, excrements, plants, pastures, and fomites); 2 -Biofilm may support virions persistence through matrix physicochemical protection against stresses such as desiccation in the extracellular medium, against immune responses (immunological modulation) and resistance to chemical substances, keeping viral particles infective over time; Surfaces can serve as a vehicle for virus dissemination because they can be contaminated by direct contact with the animal or human excretions, fluids, and through transfer by mechanical vectors (Figure 3) [START_REF] Boone | Significance of fomites in the spread of respiratory and enteric viral disease[END_REF]. The degree to which environmental/surface-based transmission of SARS-CoV-2 is actually occurring is still unclear. Considering that CoVs are enveloped viruses, it is expected a structural fragility when facing environmental conditions. However, scientists have investigated the capacity of CoVs to survive on several surfaces (plastics, latex, metals, and glasses) and the results show that they remain infectious during long periods (hours-days for CoVs, while weeks-months for others) [START_REF] Kampf | Persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents[END_REF]. In this sense, SARS-CoV, SARS-CoV-2, and MERS-CoV would be infectious in environmental reservoirs like water, foods, and in sewage time sufficiently to facilitate onward transmission. The viability and persistence of CoVs depend on several variables and the exact mechanisms are still unknown [START_REF] Zuber | COVID 19: challenges for virologists in the food industry[END_REF][START_REF] Otter | Transmission of SARS and MERS coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination[END_REF]. Although these studies consider some characteristics of the surface substrate (porous and non-porous) the biofilm condition has not been contemplated until now [START_REF] Boone | Significance of fomites in the spread of respiratory and enteric viral disease[END_REF][START_REF] Otter | Transmission of SARS and MERS coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination[END_REF][START_REF] Otter | The role played by contaminated surfaces in the transmission of nosocomial pathogens[END_REF][START_REF] Dowell | Severe acute respiratory syndrome coronavirus on hospital surfaces[END_REF]. It was shown that SARS-CoV and MERS-CoV could survive for extended periods when suspended in human secretions while the influenza virus could survive longer in mucus [START_REF] Otter | Transmission of SARS and MERS coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination[END_REF][START_REF] Thomas | Survival of influenza virus on banknotes[END_REF]. These observations contribute to biofilm-theory since organic materials are highly hydrated and rich in macromolecules which in turn can act as the extracellular matrix.

Concluding remarks

The scientific literature allows us to propose that biofilms may play a role in CoVs pathogenicity modulation, the ability to persist in reservoir hosts and the environment, and viral transmissibility. Assuming that CoVs can also occur in biofilms, new perspectives could be provided for important questions (i) the absent or low pathogenicity of CoVs within reservoir hosts, like bats; (ii) a more depressed level of

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Biofilms and viruses. Viruses are shown in the extracellular space in planktonic or biofilm forms. 1a) Some viruses come into contact with preexisting biofilms (multispecies biofilm in yellow) and adhere to them, constituting secondary colonizers, or 1b) they are enclosed in a single viral biofilm (orange). Once these structures are established, they become reservoirs and could persist like a "state of latency", keeping viral particles infective over time. 2) Biofilm detachment allows viruses to spread and can liberate virions that will colonize new sites and infect new host cells. 3a) A virion detached from multispecies or viral biofilms infecting the host cell; 3b) Viral gene exchanges and recombination events are possible in the presence of co-infections (exemplified by the green and blank viruses) and can lead to the

Figure 3 :

 3 Figure 3: Proposed role of biofilms in host-coronavirus-environmental interactions and viral dissemination. Bats are the reservoir hosts persistently infected by several viruses, potentially in the form of biofilm chronic infections (multi-species and viral biofilms depicted in enlarged view). Viruses are shed from the host into the environment in biofilms present on excretions. The environmental viral spread can contaminate organisms (e.g. humans, wildlife, livestock and pet animals, plants, and fruits) or abiotic components (e.g. fomites and water). Viral transmission is possible via direct (air-borne and droplets) or indirect (contaminated surfaces) contact between susceptible and infected individuals/hosts.

Acknowledgments

We kindly acknowledge Dr. Alexandre José Macedo, Dr. Carlos Blanco, and Dr. Daniel Thomas for their insight. We thank "Ana Paula Ilustrações e Design" and Siou Ku for the brilliant contributions with the illustrations.

Competing interests

The authors declare no competing interests.

Author contribution

Both authors contributed equally to this study.

Biographical sketch