We tested peripheral, spinal and cortical excitability during 3 minutes of unresisted finger tapping at the maximal possible rate, which induced fatigue. Subsequently, we studied the temporal dynamics of muscle fatigue, expressed in the tapping movement profile, and its relationship to neural systems using mixed model analyses. The tapping rate decreased by 40% over the duration of the task. The change in the amplitude of the range of motion was not significant. The excitability of the flexor and extensor muscles of the index finger was tested via evoked potentials obtained with various types of stimulation at various levels of the motor system. The change in spinal excitability with time was evaluated considering the simultaneous changes in muscle excitability; we also considered how spinal excitability changed over time to evaluate cortical excitability. Excitability in the flexor and extensor muscles at the different levels tested changed significantly, but similar excitability levels were observed at notably different tapping rates. Our results showed that only 33% of the decrease in the tapping rate was explained by changes in the excitability of the structures tested in the present work. Determining the central mechanisms involved in muscle fatigue is important from a physiological perspective and can also have relevant implications from an applied perspective when we refer to sports, ergonomics or certain pathological conditions.8.These mechanisms have been studied thoroughly in the case of isometric muscle contractions; they include changes in excitability both at the spinal cord and M1 networks10, which is a limitation because the CNS recovers very quickly when the activity ends12. Several works recently tested fatigue at the central level immediately following the end of unresisted RRMs without allowing time for CNS recovery14. The reduction in the maximal movement rate was greater after 30 s of finger tapping (ft) than that after 10 s, and the reduction was accompanied by an increase in the excitability of M1 GABAb interneurons, which was more pronounced after 30 s of performing the task13. Interestingly, spinal excitability, which was tested by measuring cervicomedullary evoked potentials (CMEPs), increased during the waning of the tapping rate14, which is essentially different from the outcome when fatigue is caused by isometric exercise performed for the same duration and executed with the same body segment14.Another type of muscle activity corresponding to the contractions performed during rhythmic repetitive movements (RRMs) is essential in daily living and may result in fatigue. Traditionally, their central expressions of fatigue have been studied at the point at which an activity has been completed17. Another un-resolved point is whether the changes in excitability along fatiguing RRMs are explicative for the changes observed at the kinematics of the movement.However, the description of how corticomuscular excitability changes with RRM fatigue development has not been performed, as previously done for isometric activitiespaired-pulse TMS). In another session in which the same subjects participated, CMEPs were induced by single-pulse TMS at the cervicomedullary junction level, and compound muscle action potentials (CMAPs) were obtained by percutaneous electrical stimulation of Erb’s point. Potentials were acquired while the subjects performed 3 minutes of ft at the maximum possible rate. Each subject completed two sessions, and the changes in ft during the task in the two sessions were compared.In this work, we have evaluated the temporal-dynamic changes in muscle fatigue during maximal rate finger tapping. We recorded motor evoked potentials (MEPs) “in hand muscles induced” by single-pulse transcranial magnetic stimulation (TMS) over the contralateral M1. We also conditioned this MEP (MEPc) using a previous pulse . The four participants who withdrew from the study refused to participate in the session that included CMAP and CMEP testing due to the discomfort produced by these types of stimulation. The data obtained from these participants were not included in the data analyses. The two sessions, which occurred in a randomized order across the participants, were identical except for the type of stimulation received. None of the participants took drugs or performed strenuous physical labour during the week prior to the experimental sessions. The study was approved by the Ethics Committee of the University of A Coruña. The methods used in this study conformed to the principles set forth in the Declaration of Helsinki, and all of the participants signed informed consent forms.ft at their maximal possible rate from the very beginning to the end of the 3-min task while receiving verbal encouragement. The instructions given to the subjects were “Tap with the index finger at your maximal possible rate from the very beginning and along the whole task, which lasts 3 min”. No instructions regarding the range of motion (ROM) amplitude of the movement were provided.The participants executed 13. Ft was executed on a force sensor  while index finger movement was monitored with an electrogoniometer (Biometrics S100). A Biometrics K800 unit amplified the signals from both sensors and sent them to a CED1401 (unit-1). Signal 6.0 software  sampled the recordings at 10 KHz and stored them in a computer for off-line analysis.Each participant’s dominant hand was firmly but comfortably fixed to a 3D hand-fixation structure with the thumb in abduction Fig. 13. Ft waThe signal from the goniometer was also sent to another CED1401  controlled by Sequencer . The sequencer used an algorithm to trigger stimulation at specific points in the tapping cycle (see below for further details). These triggers were also sent to the CED1401 unit-1 to determine the timing of stimulation for posterior signal analysis.first dorsal interosseous, extensor indicis, and flexor digitorum superficialis  muscles. The FDI and EXT are the specific flexor and extensor muscles, respectively, of the index finger. Prior to protocol execution, the subjects performed active extension of the index metacarpophalangeal joint to determine their maximal active ROM.Surface EMG recordings were acquired with a Digitimer D360  and sent to CE1401 unit-1. Three different hand muscles were monitored: the ft, the stimulus intensity was increased stepwise to determine the intensity at which the M-waves stopped increasing in all the tested muscles. During the task, we used an intensity 50% greater than the supramaximal intensity for stimulation. The intensities used ranged from 75 to 160 mA.We evaluated neuromuscular transmission and muscle excitability via CMAPs by stimulating Erb’s point . The cathode was placed on Erb’s point in the supraclavicular fossa, and the anode was placed over the acromion. At rest before 2 magnetic stimulator connected to a 110 double-cone coil was used to test spinal excitability. The centre of the coil was placed over the inion , and the direction of the monophasic-pulse current flowed down in the coil. The stimulus intensity was adjusted to induce CMEPs ≈5–10% of the CMAP size obtained at rest prior to execution of the protocol  placed over the hot-spot of the FDI muscle. The hot-spot was defined as the coil position at which the largest and more consistent MEPs were evoked at rest on the FDI muscle, before the fatiguing protocol. This was tested by delivering single TMS pulses at suprathreshold intensity. This position on the scalp was marked with a pen for monitoring coil placement during the session. For testing excitability during FT, the stimulus strength was adjusted to achieve MEPs ≈5–10% of the CMAP size obtained at rest prior to execution of the protocol. A paired-pulse paradigm was also used to assess short intracortical inhibition. With the same system, we tested M1 short intracortical inhibition using a subthreshold conditioning stimulus preceding by 2 ms) the aforementioned single-pulse MEP. This inter-stimulus interval is suggested to reflect GABA ms the aRecorded potentials were acquired in two different sessions. One session tested CMAPs and CMEPs, and the other session tested MEPs and MEPc values; the order of the sessions was counterbalanced across participants. During individual sessions, the two types of recorded signals were alternated in their acquisition.baseline). The participants were then prompted by an auditory cue to begin ft.We began each session by recording 10 potentials of each type at rest  and determines when the finger stops moving (the start of the contact phase). When the sequence of the first and second conditions was detected, stimulation was delivered.During execution of the Thus, stimulation was applied in all cases with the finger in the same relative position of the tapping cycle during the stationary phase when the angular velocity is zero. After one stimulus was delivered, the sequencer stopped calculating slopes from the goniometer for 4 seconds to restrict the frequency of stimulation, which can modulate excitability. At the end of the four-second period, the sequencer re-started the calculation as described above and subsequently applied stimulation again; the second stimulation always involved a different modality. Notably, however, the start of the contact phase did not occur immediately after the 4-s time lag. For this reason and because we had programmed 45 triggers during the protocol, the duration of the task was slightly longer than three minutes.ft started with MEP testing in half of the subjects and with MEPc testing in the other half of the subjects. Similar alternation in the order of testing was used in the CMAP-CMEP sessions.In the MEP-MEPc sessions, the stimulation during 20.In a time window of 2 s immediately prior to the TTL ; the obtained values are shown in Table baseline score, which applied to the CMAPs, CMEPs and MEPs but not the MEPc value; the MEPc value during the task was normalized to the unconditioned MEP during baseline.For the potentials, we first calculated the median of the 10 events acquired at rest prior to execution of the task , event #1 was considered equal to event #2, and event #45 was considered equal to event #44. We proceeded in a similar manner for CMEP and CMAP imputations.Statistical analyses of the aforementioned variables, as well as of the CMEP scores relative to the CMAP scores (the CMEP/CMAP ratio), were performed, which allowed us to estimate changes in spinal excitability considering the changes that occurred at the same time in muscle excitability and neuromuscular transmission. In the same manner, we calculated the MEP/CMEP and MEPc/MEP ratios. Also, the RMS of the EMG voluntary activity was made relative to the CMAP-RMS.task progression (in time) of the ft rate, the ROM amplitude, and excitability scores. For the excitability scores, we considered the RMS of the potentials  and the ratios mentioned above. Accordingly, we fitted fourth-order polynomials in which the subjects were modelled as random effects.Linear mixed-effects models were used to describe the ft profiles (the ft rate and ft ROM amplitude) and changes in excitability over time. Therefore, a linear mixed-effects model was used to explain changes in ft frequency and ROM amplitude as a function of time and excitability , with random effects on the subject. “Linear and quadratic functions were explored”:i is the ft profile (the frequency or ROM amplitude), Xi is the excitability score , Ti is the time (TLL event number) in task progression, and ε indicates the residual error of the model.Finally, we examined associations between changes in ft frequency  and amplitude  in the two sessions were analysed using Student’s t-test to identify differences between sessions.The normalizing scores for Previously to the above-mentioned analyses, the variables compared using a t-test and the residuals of the nonlinear mixed effects models passed the normality tests.nlme package). Based on previous recommendations23, we set p < 0.01 as the level of significance due to the relatively small sample size of our study.We performed statistical analyses using R software  used to normalize the scores during task execution. The intensity of TMS (for MEP and CMEP) was selected to obtain potentials of ≈5–10% of the CMAP size in the corresponding muscle; the stimulation parameters in both sessions are showed in Table Table 9 = 0.75, p = 0.4) .The maximal active finger index extension ROM (averaged across participants) acquired before execution of the 3-minute task was 50.6° (SEM = 3.3) for session 1 and 51.9° SEM = 2.3) for session 2; these values were not significantly different (t.3 for seft profile did not differ in either frequency (p > 0.2) or ROM amplitude (p > 0.2) in the two sessions. The tapping frequency changed significantly during the task (p < 0.001) in both sessions, decreasing rapidly within the first minute, decreasing more shallowly during the second minute, and reaching a plateau in the third minute  , increasing and reaching a maximum value at approximately 30 s and then decreasing progressively during the remainder of the 3- min task Fig. .Figure 4baseline (rest) value at the beginning of the task. The profile along the 3-min duration of ft is shown in Fig. The CMEP was approximately 5-times larger than the ft, and Fig. Figure baseline , the MEPc increased progressively until nearly the end of the task. When this evolution is represented relative to the changes in MEPs during the task , although the magnitude of the change was small Fig. .Figure 5ft execution (p < 0.001); when ft began, spinal excitability was 5-times greater than the excitability at rest and increased further to reach its peak after 30 s. It then decreased progressively during the 2nd minute and remained stable (approximately 5-times larger than that at rest) during the final minute.The evolution of the CMEPs during the task is shown in Fig. The change in MEPs Fig.  is shownbaseline was smaller than the MEP; the conditioning pulse reduced the RMS by ≈20%   Fig. .Figure 6ft was small in magnitude but statistically significant (p < 0.001)The profile of spinal excitability  is shown in Fig. ft execution, and the MEPc became larger than the MEP  but were smaller in magnitude. Remarkably, again, the MEPc at rest was smaller than the MEP on average Table , but thiMEP Fig. . The cha ft Fig.  were sigf of Figs. Stimulation was applied during the contact phase of the tapping cycle. At this phase the voluntary EMG activity -RMS-, made relative to the changes in the RMS of the CMAP at the same time, changed significantly during task execution for the FDI, EXT, and FDS . However, in the three muscles, the effect was small in magnitude , spinal excitability (CMEP/CMAP) and cortical excitability (MEP/CMEP and MEPc/MEP) explain changes in ft frequency (Table The excitability of the FDI muscle (a flexor muscle) explained the changes in cy Table . Figure cy Table . Compareft begins. At this time, the ft rate was ≈80% of the maximum rate . The ft ROM amplitude did not change significantly. At this point, we must consider that during a rhythmic repetitive movement, a close inverse relationship exists between the tapping rate and ROM amplitude. Tapping at the maximal possible rate causes a reduced ROM amplitude; therefore, if participants were to tap with a maximal ROM, their maximal ft frequency would be much lower. In this case, the behaviour of the flexor and extensor muscles should probably be different, as would also be the case if the movement were performed against resistance. This is a key point that must be considered to properly understand the scope of our study: the particular relationship between rate and ROM amplitude was observed chiefly because subjects performed maximal-rate ft with a very small ROM amplitude .The execution of ft profile is consistent with that found in previous studies in which similar tasks were evaluated. Performing tapping at the maximum possible rate for 30 s has been shown to reduce movement frequency by approximately 15–20%14, as in the present work. The temporal evolution of ft ROM showed changes that were less pronounced than the changes in the ft rate, which is consistent with previous works that used tapping tasks of much shorter durations14. As previously mentioned, since tapping at the highest rate is bound to produce a smaller ROM, the fatigue related to the tapping rate may not necessary be similarly linked to ROM.The behaviour in the Interestingly, the changes in the excitability of the flexor and extensor muscles during task execution were significant despite the antagonistic functions of these muscles. The extensor works against gravity to lift the finger, and the flexors act to stop extension and perform flexion  and in a specific index finger flexor . Spinal excitability in the flexor digitorum superficialis was modulated to a lesser extent during task execution. Notably, this muscle does not act specifically on the index finger but also acts on other fingers. As an aside, in our experimental setup, the wrist was fixed in extension, which may contribute to the inhibition of this muscle at presynaptic and postsynaptic levels within the spinal cord25. For this reason, the change in excitability that occurs in this muscle during the task, as well as the association between changes in excitability and the ft profile (as discussed below), may be quite different from the changes that occur in the other two explored muscles. In the FDI and EXT, spinal excitability changed significantly with time, but in both cases, it remained well above the level of baseline excitability tested at rest, and the deviation from those elevated levels of excitability was small.In contrast to the evolution of the CMAP, spinal excitability  exhibited a large change during task execution. Spinal cord excitability was approximately 5-times larger at the beginning of task execution than that at rest both in a specific index finger extensor . Our protocol controls for these sources of bias. Some other studies in which these limitations were controlled have consistently shown increased excitability of inhibitory interneurons (GABAb) during fatiguing ft5. We did not specifically test those circuits in the present work, but the current results suggest that the increased excitability of inhibitory GABAb -interneurons in M113 is likely compensated for by changes in the excitability of some other neural population, as shown by the results of the MEP/CMEP ratio.Cortical excitability, as measured by the MEP/CMEP ratio, changed significantly during the task in the three muscles. Notably, the magnitude of this change differed in the three muscles. In the two muscles that act specifically on the index finger (FDI and EXT), excitability increased during the 3-min task. In the FDS, the small but significant increase in excitability reached a maximum at approximately 90 s, and excitability then decreased slowly. In light of previous work that has shown a clear increase in cortical excitability, our results were to some extent unexpecteda inhibition in M126. Remarkably, a standard paired-pulse protocol for testing this form of inhibition  produces a very powerful inhibition on the conditioned MEPs; thus, this methodology might impede the detection of increased levels of inhibition generated by the task. To avoid this, at baseline, we set individualized conditioning pulse intensities producing conditioned MEPs smaller the un-conditioned ones, but larger enough to permit its modulation by the execution of the fatiguing task. Unexpectedly, however, during task execution the effect of the conditioning pulse on the MEP was the opposite: it produced facilitation of M1 in all the tested muscles. We have not identified the physiological mechanism that underlies this observation, but it may involve changes in the excitatory/inhibitory balance of M1 during the execution of the fatiguing task. The parameters that we used at rest were selected to permit evaluation of intracortical GABAa inhibition26. Interestingly, intracortical excitatory circuits can also be tested using similar ISI28. This is the case for short intracortical facilitation (SICF), where a subthreshold pulse (delivered 1–5 ms after a suprathreshold pulse) increases the MEP size, an effect that is mediated by M1 excitatory interneurons29. One may be tempted to speculate that in our experiment, a similar mechanism may be operating in which the conditioning pulse arrives at a moment at which altered excitatory/inhibitory balance is produced by the task. On the other hand, although our results cannot explain the mechanism underlying this observation, a very relevant implication of these results is the need to test fatigue expressions at the time of fatigue14 as the excitatory/inhibitory balance at that time is quite different from the balance at rest30.We evaluated some of the possible compensatory circuits using paired-pulse TMS with an ISI of 2 ms, likely testing GABAβ1 positive and β2 negative 13; in that case, a slowing of muscle relaxation was evident at the end of the task in the presence of a tapping rate decrease of ≈15%. This mechanism appears to be much less relevant in the case of isometric fatiguing tasks13. In the present work, we observed decreases in tapping rate of up to 40% at the end of the task; however, the changes in the cortical excitability (MEP/CMEP) of the FDI muscle, which was the parameter with the greatest explanatory value, accounted for ft rate reductions of only up to 13% .Despite the above-described clear and significant changes in excitability, our study was designed to answer a more relevant question: Do these changes in excitability explain the modifications in tapping rate or amplitude? To answer this question we tested the association between the changes in tapping profiles and excitability. In some muscles and variables only linear functions were significant. Thus, the behaviour was well explained by a lineal fit, the change in the tapping profile is therefore constant with the change in excitability  has to be considered during fatigue development32.Apart from peripheral elements, central mechanisms can also coexist. At this point, one must keep in mind that our work explored an important but limited number of central circuits with potential relevance to fatigue. Other populations of interneurons (inhibitory or excitatory) may play a role in the development of fatigue. This appears to be the case for inhibitory GABA16. Following this line of thought, the central mechanism of fatigue during RRM might be linked to the precise sequence of activation of antagonistic muscles33. However, as far as the corticomuscular axis is concerned, the excitability of the structures explored in our work appears to have a limited impact on the inability of the subjects to maintain the maximal tapping rate during repetitive movement.Alternatively, the presence of central adaptations during fatigue might not have a correlate in excitability changes reflected by the MEP. For instance, the reduction in muscle force and central drive to the muscle might not match changes in the MEP features16, the use of larger samples would allow a more confident estimation of the findings. To account for this limitation, we adopted a conservative approach in our data analyses, and the level of significance was set at p < 0.01, as has been recently recommended23.Several points should be considered limitations of our study. First, the study sample size was small. Four of the 14 initially recruited subjects chose not to take part in the CMAP-CMEP session due to the discomfort produced by these techniques. Thus, only 10 participants completed the two sessions. Although similar sample sizes have been used in relevant studies in the fieldft in our study was anti-gravitational, and the flexor muscles worked in favour of gravity. Since the movement was not resisted and the mass of the index finger is small , we have discussed our results as stemming from an unresisted movement, but it is true that the resistance for the extensors and the flexors in our task is not exactly the same. This is important since repetitive movements against higher levels of resistance might present different evolution of muscle excitability and processes related to muscle force production34.Second, the extension phase of hot-spot of the FDI, which has a main role in this task24, and with this hot-spot we recorded potentials in other two muscles. We adopted this approach since the cortical spatial distribution of neurons targeting hand muscles is overlapped. This has been shown previously by different image studies in human36 and with neuronal recordings in monkey37. Therefore, in our study, even though we were on the hot-spot of the FDI, we were expecting to stimulate a large proportion of the neuronal pool targeting the other two muscles engaged in digital movements. In fact, as observed in our work (Table hot-spot is suitable for our purpose.Finally, during the recording of the MEP and MEPc, we focussed on the cortical rk Table , the MEPOur work shows that changes in excitability along the corticomuscular axis play a role in the fatigue that develops during unresisted repetitive movements but cannot fully explain the observed decrease in the tapping rate. When muscles that act specifically on the index finger were tested, increased spinal excitability in both the flexor and the extensor was associated with a lower tapping rate. The cortical excitability of the specific flexor muscle tested, which plays a main role in the rapid change from extension to flexion that is necessary to produce higher tapping rates, increased with higher tapping rates.


The working conditions in a slaughterhouse are difficult because of the low temperatures, high humidity, and little natural light. Therefore, in these facilities, there is a high demand in the maintenance of strict hygiene rules. Lately, the new SARS-CoV-2 pandemic situation has brought new challenges in the meat industry, as this sector has to maintain its operability to supply the meat and meat products demanded by the consumers. In this challenging period, the safety of the workers is as important as keeping the high demands for the safety of the meat and meat products along with consumer confidence. This paper aims to give an overview of the risks associated with the SARS-CoV-2 virus transmission between the workers in slaughterhouses and to evaluate the stability and infectivity in the working environment of these facilities. Considering the persistence of this virus on different surfaces and the environmental conditions affecting its stability , in the study we proposed several short-, medium-, and long-term preventive measures for minimizing the potential threats of the actual pandemic. Meat and meat product consumption has registered an increasing trend since the early 1960s , and shoEven though the consumption of proteins from plant-based products has been maintained stably in the last 60 years, meat and meat products are the major source of proteins, minerals, and vitamins , and repNowadays, the meat industry in the European Union comprises over 32,000 companies with over one million employees. This industry represents 1.53% of the gross domestic product (GDP) .Such a high demand for meat and meat products leads to intense activity in slaughterhouses around the world. The type of slaughtered animal differs between countries as a consequence of wealth and culture .Generally, because of the different services that are taking place in the slaughterhouses, there is a continuous need for facilities and equipment that assure a safe production of meat and meat products designed for human consumption, along with the safety of the workers. In general, a slaughterhouse can encounter all types of contaminants, physical, chemical, and biological. The growth of pathogens is influenced by how the animals are slaughtered, eviscerated, and stored, and therefore, the potential of contamination of the meat and meat production is high . The preMeanwhile, because of the continuous contact of the workers with biological materials, they can be exposed to zoonotic pathogens , a fact By the end of 2019, a COVID-19 outbreak emerged in Wuhan, China, and the World Health Organization (WHO) declared it a pandemic in February 2020. Some authors have considered that, because of the dynamics of transmission of the SARS-CoV-2 virus, this should be assimilated to a zoonotic illness ,12.Considering the congestion of the workers in the slaughterhouses, any possible zoonotic outbreak will be worsened, and the application of the existing bio-risk mitigation measures could be a challenge.This paper focuses on the evaluation of the risks associated with potential infection with COVID-19 of slaughterhouse workers. The study considers the working environment and most used inanimate surfaces during the activities in these facilities. Moreover, some measures for the safety of the workers are proposed.For the present paper, we carried out a critical literature review, and the most relevant publications were retrieved from different journal databases. The search of the literature focused on papers published in ScienceDirect, SpringerLink, and ProQuest Central databases. The authors considered articles published from the beginning to the end of 2020, including the available accepted journal papers planned for volumes with appearance in 2021. The following keywords were selected and used with Boolean operators: slaughterhouse, SARS-COV-2, COVID-19, zoonosis, worker safety, risks, inanimate surfaces, persistence, and environmental conditions. The present study excluded, although consulted, the manuscripts present in the preprints server that are figured as preliminary reports and have not been peer-reviewed. The investigators evaluated the screened material and considered the most appropriate information regarding the workers’ safety in slaughterhouses. Considering the structure of the present paper, the number of consulted references is assigned as follows: (i) 13 references for zoonotic pathogens; (ii) 25 papers for the stability of the SARS-CoV-2 virus in the environment; (iii) 14 references for the environmental factors that favor the transmission of pathogens in slaughterhouses; and (iv) 11 articles for the proposed safety measures.Zoonosis is referred to as infectious diseases that are provoked by a pathogen that is transmitted from an animal to a human. Zoonotic pathogens can comprise bacteria, viruses, or parasites that can be spread to humans through food products, water, or the environment. Some of the zoonotic viruses that can be transmitted to slaughterhouse workers refer to viruses that provoke diseases, such as contagious ecthyma, influenza, rotavirus, Newcastle disease, cowpox, and Louping ill . These vCoronaviridae that is further divided into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, however, it was found that only alpha- and betacoronaviruses are infectious for humans [Besides the viruses mentioned above, there is also the class of r humans ,16. Coror humans ,18,19,20Up until now, the most known outbreak provoked by the coronaviruses was in China in 2002, the severe acute respiratory syndrome coronavirus (SARS-CoV), and this spread to 33 countries ; then, iSince the end of 2019, a new strain of the severe acute respiratory coronavirus, SARS-CoV-2, also named COVID-19, became a health emergency that is of international concern . The COVThe novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belongs to the genus Betacoronavirus, and is an RNA virus ,26,27,28The transmittance of the virus can be through direct contact between the infected patient and a healthy person, through sneeze or cough respiratory droplets, and from contaminated surfaces or environments ,33. The safety of the workers of slaughterhouses is always a challenge, but nowadays, because of the new COVID-19 pandemic situation, this needs special attention because of the environmental working conditions. These working conditions refer mainly to the high humidity associated with the amount of water used for washing the carcasses, equipment, and surfaces, low temperatures, and the presence of residual organic material ,8,9,34.Another issue that should be considered is that, working at temperatures between 4 and 10 °C, the breath of the workers will produce condensation of the exhaled air and rapid humidification of the protective masks, a fact that will contribute to an inappropriate filtration of the air or even to inappropriate wearing of the protection equipment.The attachment of the virus on inanimate surfaces is dependent on the physicochemical properties of the surface ,36,37. VRecently, several papers have been published containing information about the persistence of coronaviruses, and the researchers concluded that human coronaviruses could persist on different surfaces for up to nine days, but, fortunately, a thorough disinfection procedure would lead to virus inactivation ,50,51,52Van Doremalen et al.  showed tThe studies of the influence of temperature and relative humidity on the stability of the COVID-19 virus on non-porous surfaces indicated a rapid decay of the viral load if either temperature or humidity increased . MoreoveThe persistence of the COVID-19 virus in aerosols is another aspect that should be considered. While in one study  it was rSpecial attention should also be paid to the presence of the SARS-CoV-2 virus in wastewaters, as the virus was also detected in feces and urine ,56,57. BLike all of the other cold food processing establishments, slaughterhouses and meat processing factories are considered as vectors for the spread of COVID-19. According to the literature, the major causes that are contributing to a higher risk of virus transmission in the meat sector are the microclimates in the abattoirs, materials used for the equipment inside of the slaughterhouses, the type of ventilation of the air, organization of the workstations, the type of equipment used, the ability of the workers to respect the safety rules, the awareness of the workers in respecting the prevention measures in case of infection with the COVID-19 virus, the assurance of transportation in safe conditions, and respecting the minimum recommended distance between individuals.10 after 14 days. On the other hand, when the incubation temperature was increased to 70 °C, the inactivation times were reduced to only 5 min [Meat plants’ microclimate  are favorable for coronavirus to thrive and spread . Experimly 5 min . In anotly 5 min  evaluately 5 min , while oly 5 min .Metallic and plastic surfaces maintain live viruses for a longer time than other materials. At an ambient temperature (22 °C) and 65% relative humidity, no infectious virus could be detected on stainless steel after seven days, on glass after five days, on wood and textiles after two days, and on tissue paper after 3 h . AccordiAir conditioning systems in slaughterhouses may spread virus particles and therefore increase the risk of infection. The information about the persistence of SARS-CoV-2 in the air is known only from hospital environments. Air sample analysis from a COVID-19 ward at a hospital in Wuhan, China indicated the presence of virus RNA in the air even at 4 m from patients . In anotIt is well-known that the equipment in slaughterhouses is noisy, i.e. grinders, mixers, tumblers, bone saws, and crushers, and these require workers to talk loudly, increasing the risk of spreading virus-infected droplets. Moreover, the specific working conditions in proximity to one another, with increasing line speeds and the internal circulation in these plants, make it difficult to keep a safe distance between the workers, and certainly contributes to the spread of the virus. In most European countries, the meat industry depends on migrant and cross-border workers. Because of the need to save as much as possible from their salaries, these workers share rented dormitories with several other people, and most of the time, these facilities do not meet the minimal hygiene standards. Therefore, respecting social distancing in these accommodations is almost impossible. Moreover, the workers are transported together on buses, without ensuring social distancing during transport. Another aspect that is contributing to the virus spreading between workers is that, in the case of COVID-19 symptoms, most of them do not report their health status and continue to go to work because of the fear of losing their job.In meat factories, cleaning is performed by high-pressure washing of floors, surfaces, and equipment. These methods produce large amounts of droplets and generate aerosols. If the floor and surfaces are contaminated by infected workers, viruses will be spread into the air, and could be a potential risk of infection for other workers .Another problem frequently met in slaughterhouses with a big number of foreign employees is the difficulty of implementing biosecurity mitigation measures that can be respected by a multicultural working community that might not speak and/or understand the native language spoken in the host country.The preventive health and safety measures adopted to protect workers are also affected by the decreased frequency of labor inspections during the pandemic period.A rigorous mitigation action plan is necessary, as the vulnerability to natural, biological, chemical, or any other types of hazards might not be prevented through the existing public health risk mitigation measures . According to some public health authorities, like the Centers for Disease Control and Prevention (CDC), World Health Organization (WHO), and Food and Drug Administration (FDA), there is no evidence for severe acute respiratory syndrome coronavirus (SARS-CoV-2) direct contamination or transmission by the ingestion of contaminated food ,64. However, it cannot be ignored that there is a possibility of spreading the novel coronavirus through contaminated food or food packages, as there are insufficient and edifying studies reporting on this subject . FurtherRecently, some authors have reported on foods and food packaging materials contaminated with SARS-CoV-2. Harbourt and collaborators reported that SARS-CoV-2 remained largely viable on pork skin after 14 days at 4 °C and four days at 22 °C with a relative humidity of 40–50% . BecauseIn another study, Fisher et al. demonstrated in their laboratory experiment the survival ability of SARS-CoV-2 at temperatures and time associated with refrigerated (4 °C) and frozen food (−20 °C) storage and transportation conditions. On pork, salmon, and chicken samples inoculated with SARS-CoV-2, the viruses maintained their infectivity even after 21 days for both refrigerated and frozen samples .In June 2020, in one of the Beijing agricultural product markets, SARS-CoV-2 was identified on a cutting board used for processing imported salmon. Not much longer after that, in July 2020, the coronavirus was detected on the packaging materials of frozen shrimp imported from Ecuador. In August, local authorities of Guangdong confirmed the presence of SARS-CoV-2 on frozen chicken surfaces imported from Brazil . The detection of SARS-CoV-2 on frozen foods suggests that these cases are not random, isolated incidents, but rather are alarming signs that viral contamination and foodborne transmission may present a systematic risk in the ongoing pandemic. Although there is no certainty of food transmission in slaughterhouses and meat factories, due to the large number of infestations reported among workers in this sector of the food industry, it is necessary to take safety measures regarding food contamination from infected people. Food handlers infected with COVID-19 can expel droplets by breathing, talking, coughing, and sneezing, contaminating the foods or the packaging materials in proximity . AdditioThe meat processing sector, as well as the entire food industry, needs to prepare for epidemics and pandemics with new technological solutions, including a greater degree of automation and robotics, to reduce human food contact and the human density in workplaces to minimize food waste and ensure food safety .The plan for risk minimization of COVID-19 in the meat industry should consider two directions, (i) in terms of food safety for the prevention of contamination of susceptible food or packaging materials and (ii) concerning the employees’ safety to ensure safe working conditions .For the safety of the workers in slaughterhouses, we are proposing short-, medium-, and long-term measures. Regarding the short-term, special attention should be paid to the (i) identification and isolation of contaminated persons; (ii) periodic training of workers on virus spreading, the correct methods of prevention and sanitation, and the importance of wearing protective equipment; (iii) active symptoms screening, including temperature monitoring of all workers and visitors; (iv) using adequate personal protective equipment (PPE); (v) strict observance of effective hygiene practices; (vi) maintaining physical distancing both on the production line and during breaks; and (vii) the use of appropriate and effective disinfectants.The medium- and long-term measures should focus on (i) fast and efficient methods of identifying contaminated persons or areas; (ii) increasing the degree of automation and robotization of technological processes; (iii) finding effective sanitation alternatives that do not affect the health of workers and/or pollute the environment; and (iv) finding technological solutions, such as biosensors or nanosensors, to identify the virus on equipment, workspaces, and surfaces of the food or food packaging.Based on the findings already published in the literature, slaughterhouse managers should consider more rigorous preventive measures implemented in their safety management systems for combating the SARS-CoV-2 virus, and a good start could be from the measures proposed in Special attention should be paid to the workers’ awareness and hygiene, along with a thorough check of their health status to maintain the safety of all workers. An important aspect is to apply a good communication procedure to assure that all of the workers have understood the application of the safety measures and that the language hindrances are overcome. Not of less importance is the necessity to use more frequently the disinfectants appropriate for use in the meat industry, and disinfect more often, especially surfaces that are proven to maintain the viability of the COVID-19 virus for a longer time.Last but not least, special regulations should be adopted for the encouragement of the workers to declare any symptoms of illness and self-isolate. This should be sustained by an adequate payment that will dispel the fear of unemployment.


Viruses are tiny particles that cannot replicate – or survive for very long – outside the cells of their host organism; yet they remain an ongoing risk to our health and our eye health.Globally, viral infections of the eye are common. They are a significant cause of acute red eye and visual loss. Any part of the eye and adnexa – from the eyelids to the retina and optic nerve – can be affected by viral disease. Some ocular viral infections, such as viral conjunctivitis due to adenovirus or influenza virus, are short lived, with limited ocular complications. However, other viral infections can cause serious complications, such as corneal scarring from stromal keratitis due to herpes simplex virus (HSV) or retinal detachment resulting from cytomegalovirus (CMV) retinitis.Certain viral eye diseases demonstrate clear clinical signs that enable diagnosis . In other cases (for example follicular conjunctivitis or uveitis), a variety of different viral infections may be responsible for the same clinical signs. Making a specific diagnosis can therefore be very challenging.Antibiotics are not effective against viruses, but evidence-based anti-viral treatments exist for several viral infections, including herpes simplex (HSV), varicella zoster (VZV), cytomegalovirus (CMV) and human immune-deficiency virus (HIV).In this issue, we present reviews of eye disease due to herpes simplex and herpes zoster. These are both DNA viruses (see p. 67) and tend to lie dormant in nerve ganglia. Adenovirus is another DNA virus; it usually causes an infectious follicular conjunctivitis. There are also review articles on viral diseases which have come into more prominence in the last two to three decades: HIV, CMV and, more recently, Ebola and Zika. Finally, we have included an article that reviews our current understanding of the available treatments for ocular viral infections.The aim of this issue is to present an overview of the common and important ocular viral infections, how a clinical diagnosis can be made, and the steps that can be taken to prevent and treat ocular viral infections in order to reduce eye disease and visual loss. If you find it useful, please share it with your colleagues and team members.Viruses are very small infectious agents . They coGenetic material (RNA or DNA)A protein coat (the capsid) that surrounds or protects the genetic materialA lipid envelope around the capsid, studded with unique surface proteins (glycoproteins). Some viruses do not have an envelope.Viruses are very simple organisms and can only multiply within the living cells of a host organism, such as a human being. A virus will enter the cell and trick it into making copies of the virus until the cell bursts and releases the copies, often destroying the cell in the process.The viruses – known as viral particles or virions when outside a cell – can then invade other cells or be transmitted to another host. The main mechanisms of transmission are:Exchange of bodily fluids Inhalation of airborne viral particles (after an infected person coughs or sneezes)Transfer of viral particles from contaminated surfaces to the eyes, nose or mouth, usually via the hands. Surfaces – including ophthalmic equipment – can become contaminated when an infected person coughs or sneezes, or touches the surface after touching their nose, mouth or eyesContaminated surgical instruments that come into contact with body tissues and fluids during surgery.Viruses cause disease in two ways:The direct action of the virus on cells. For example, herpes simplex dendritic ulcer.The body's immune response to the virus and/or virus-infected cells. For example, herpes simplex disciform endotheliitis (p. 69).The body's immune system responds to a viral infection by making specific antibodies (the immunoglobulins IgM and IgG) designed to bind either to the unique viral surface proteins, or to viral fragments that remain outside the host cell. The antibody can help to mark the virus or infected cell for destruction by the body's T lymphocyte cells . Once the viral infection has been halted, the memory to make antibodies remains in the immune system of the host, which allows the immune system to recognise the same virus immediately and act to stop a new infection.Immunisation takes advantage of this fact. It involves exposing the host to a non-pathogenic version of the virus: so that the person doesn't become ill, but the immune system produces antibodies against future exposure to a pathogenic version of the virus. This approach is used successfully with some viruses, such as measles and rubella, which are considered ‘stable’. However, with other viruses, such as HIV, the surface proteins are continually changing, so that antibodies do not work effectively. The immune system struggles to fight such virus infections and immunisation is therefore problematic.Some viral infections can also be prevented by reducing transmission between people. Avoiding unprotected sexual intercourse reduces the risk of transmission of human immunodeficiency virus (HIV) and herpes simplex virus 1 (HSV-1). Regular and effective handwashing helps to prevent the transmission of some viruses e.g., adenovirus.In a hospital or clinic setting, you can reduce transmission between doctor and patient, and from patient to patient, by practising good hygiene. This includes:Close attention to handwashing (scrubbing)Cleaning of all surfaces, equipment and instruments using appropriate methods and antiviral solutionsWearing protective clothing such as gloves and, in some circumstances, a mask.Editorial team


The costs attributable to antimicrobial resistance (AMR) remain theoretical and largely unspecified. Current figures fail to capture the full health and economic burden caused by AMR across human, animal, and environmental health; historically many studies have considered only direct costs associated with human infection from a hospital perspective, primarily from high-income countries. The Global Antimicrobial Resistance Platform for ONE-Burden Estimates (GAP-ON€) network has developed a framework to help guide AMR costing exercises in any part of the world as a first step towards more comprehensive analyses for comparing AMR interventions at the local level as well as more harmonized analyses for quantifying the full economic burden attributable to AMR at the global level.GAP-ON€  is composed of 19 international networks and institutions active in the field of AMR. For this project, the Network operated by means of Delphi rounds, teleconferences and face-to-face meetings. The resulting costing framework takes a bottom-up approach to incorporate all relevant costs imposed by an AMR bacterial microbe in a patient, in an animal, or in the environment up through to the societal level.The framework itemizes the epidemiological data as well as the direct and indirect cost components needed to build a realistic cost picture for AMR. While the framework lists a large number of relevant pathogens for which this framework could be used to explore the costs, the framework is sufficiently generic to facilitate the costing of other resistant pathogens, including those of other aetiologies.In order to conduct cost-effectiveness analyses to choose amongst different AMR-related interventions at local level, the costing of AMR should be done according to local epidemiological priorities and local health service norms. Yet the use of a common framework across settings allows for the results of such studies to contribute to cumulative estimates that can serve as the basis of broader policy decisions at the international level such as how to steer R&D funding and how to prioritize AMR amongst other issues. Indeed, it is only by building a realistic cost picture that we can make informed decisions on how best to tackle major health threats. In recent years, numerous global institutions including the United Nations , World HWhile older studies focussed on costs to health services, more recent studies have included many other costs that AMR imposes on society more widely. These costs derive from the prolongation of illness and increased levels of mortality, work absenteeism and reduced labour efficiency, disruption in international trade, reduced livestock production .The more publicised estimates of the cost of AMR have been at the macro-economic level, with forecasts far into the future, using extrapolation and projection from mathematical modelling. The results of these studies have been staggering. The impact of AMR over the years until 2050 is estimated by the World Bank to be within the same order of magnitude as that of the major 2008 global financial crisis . The estWhile useful for giving a sense of the scale and high cost of inaction globally, these are rough, top-down estimates, based on numerous parameters with significant data gaps and thus requiring many assumptions. Such estimates are of limited use for understanding the full cost of AMR at the local level or for making decisions about how to tackle AMR in an economically optimal manner. The nature of the current data also hinders our ability to make appropriate trade-offs between policy options. Where estimates have been made on a micro level, they have looked mainly at costs derived from the human health burden imposed by AMR, ignoring indirect costs and costs derived from resistance in animals and the environment . This agThe GAP-ON€ network is funded through the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) 8th call as part of the Virtual Research Initiative 2018 . Its purExpert consultation took place across the GAP-ON€ network, which includes 19 smaller networks and institutions, comprising human and veterinary infectious diseases physicians and microbiologists, experts in AMR burden estimation, food safety, health-economics, international law, as well as infection control experts, clinical epidemiologists, statisticians, and health information librarians.The costing framework was constructed by means of teleconferences, Delphi rounds and a face-to-face meeting, to reach consensus on the perspective of the framework, the list of relevant pathogens, applicable costs, epidemiological data requirements, and data quality dimensions. Interim reports and feedback were managed through electronic exchange. A REDCap survey tool  was usedThe focus of this work is currently limited to bacterial resistance to antibiotics. We maintain the term “antimicrobial resistance” to highlight that this work can later be extended to antifungal, antiviral and antiparasitic resistance. The framework focusses on the costs associated with colonization or infection by single or multiple resistant pathogens, as opposed to its antibacterial sensitive counterpart.1For the purposes of this costing exercise, colonization was defined as the presence of bacteria from a non-sterile specimen  in a sufficiently high concentration that it can be detected, but without causing signs or symptoms of disease. Colonization can persist for days to years, and a person or animal colonized with a drug-resistant organism may transmit it to other humans/animals; it may not always impose additional costs.Infection was defined as clinically and  microbiologically confirmed disease, including identification of an invasive pathogen from a specimen combined with infection-specific symptoms; it generally imposes costs within both human and animal health settings at some level.The starting point for the selection process was the WHO list of priority antibiotic-resistant bacteria . In addiThis work includes all of primary setting in which antibiotics are used, and where there is potential for transmission of resistant bacteria (and resistance genes)  Fig. 1)1).Fig. The resulting framework consists of the epidemiological data and the cost components that need to be collected to build a credible picture of AMR-costs from the local level Fig. .Fig. 2tAMR pathogens can cause either colonization or disease in human or animal health, and can be transmitted within and between One Health areas Table .Table 1Probabilities can be estimated based on the epidemiological data collected Tables , 3 and 4The literature confirms the presence of similar strain types, clones, resistance genes and associated mobile genetic elements across human, animal and environmental health in different permutations and combinations where two or more elements of the One Health triad have been assessed . HoweverSingle drug resistanceThe costs associated with resistance depend on the antibiotic to which resistance has developed as well as other contextual factors. Some potential scenarios include the following:2.Multi-drug resistanceResistance emergence to a drug that was previously effective and widely used, but where there are equally effective and safe alternatives that are readily available; in this case the costs will mainly revolve around more expensive treatment, more diagnostics, more frequent side-effects/lower tolerability, and longer hospital admission .3.Pan-drug resistanceResistance emergence to drugs, where equally effective and safe alternatives are not readily available, but the alternative drug(s) becomes more readily available as transmission increases; here the costs need to cover all of the above, plus those associated with worsening infection, possibly more often resulting in death, acquiring a more expensive treatment, side-effects/lower tolerability .Resistance emergence to a last resort antibiotic (creating a pan-resistant pathogen); here opportunity costs with regards to avoiding high-risk treatments should be considered, the costs/burden beyond the health sector will become more pertinent, like lost productivity and trade, less tourism, expedited R&D, etc. .Direct costs associated with care in each of the relevant health care settings should be considered . Indirect costs deriving from the time away from work for patients and carers, informal care by others and their loss of productivity, should also be included. Costs related to healthcare avoidance should also be considered. Cumulatively, any loss in productivity due to absence or disabilities related to resistant infection should also be included.Direct costs related to the treatment of companion animals and animals in the food chain should be included. Ideally the effect on the quality of life of the owner of companion animals lost to resistant infections would also be considered. Indirect costs associated with resistance in food chain animals should be considered at the level of the individual farmer as well as at the sector-wide level if there is transmission that affects food chain supply . The different species of companion animals  and of food chain animals  should be considered individually.Environment here accounts for water, soil and air as well as plants and crops affected by environmental pollution with antibacterials and drug-resistant microorganisms from contaminated manure, pharmaceutical manufacturing waste, hospital waste, wastewater treatment facilities, untreated human waste, waste and runoff from aquaculture, livestock, and plant-based food production and processing facilities .Table 8While the costs associated with treating resistant infection do not generally apply to wildlife, the knock-on costs to species elimination, the undermining of ecosystems, and other related costs should be considered .AMR is not a disease, with its own recognisable signs and symptoms and clearly associated health burden and epidemiological parameters. Rather it refers to species that have acquired drug resistance mechanisms, that have replicated, and overgrown other species that can colonize or infect people, animals, or the environment causing negative effects. The emergence of resistance is often due to selective pressure of antibiotic use, or during the acquisition of DNA from other species or strains, rendering the strain capable of resisting the inhibitory activity of some antibiotic (e.g. plasmid-mediated mechanisms of resistance). An infection that has not been a threat for many decades, over time can become resistant to currently available therapies, and potentially all existing therapies, and therefore become a threat again. This change within known existing pathogens, and the fact that one pathogen may be responsible for multiple types of infections (e.g. from UTI to CNS abscesses), can make AMR difficult to understand and address within the political sphere as well as within public discourse . In addiArguably this hinders to some extent our ability to place AMR appropriately within the hierarchy of political priorities and to address it with suitable urgency. An important tool in communicating concerns about AMR is to address the resulting economic consequences in a comprehensive and more immediately relevant way .In addition, while previous rough, top-down estimates have helped ring the alarm bells within important international fora, they are insufficiently nuanced to help guide decisions. If a common, bottom-up framework such as this one can be applied in a selection of sites, we would be able to collect the necessary data to communicate the importance of AMR on the international and national agendas, including the appropriate focus on the various settings and sectors. Also, such cross-national estimates of costs within these settings and sectors can help guide research and development efforts, and appropriate funding schemes.At national and local levels, a bottom-up costing is essential for choosing the optimal way to tackle AMR. Numerous different prevention, control, and treatment measures are available to help combat AMR. However, to compare existing measures or assess the potential of new ones we need to be able to estimate the costs that such interventions will impose as well as the costs that their implementation will off-set . This bottom-up cost framework is intended to facilitate the estimation of these cost off-sets in particular, although the One Health cost “ingredients” will also cover most of the implementation costs associated with any intervention and thereby make any intervention-related costing exercise far simpler. Ultimately a bottom-up costing framework should help simplify any eventual economic analysis – whether it be cost-of-illness, cost-utility, or cost-benefit in structure.This work focusses on the additional cost of drug-resistant infections and drug-resistant colonisation, when compared to a drug-sensitive counterpart, answering the question “What is the impact of antibiotic-resistant infections (or colonization) relative to the same infection that would be susceptible to first line treatments?”. Sometimes, comparison to non-infected patients would also apply; this is particularly relevant when estimating the possible preventable burden of AMR, while focusing on an intervention that eliminates resistant as well as susceptible infections, like for example vaccination. In this scenario the question “What is the impact of antibiotic-resistant infections compared to a situation in which such infections would not occur at all, because they were prevented?” would be most relevant, which would require a comparison to no infection .When it comes to the costs associated with mortality, the ability to attribute death to resistance is particularly challenging.2For some of the components, especially at population level, attribution is fairly straightforward. For example, costs associated with enhanced resistance surveillance, consumption surveillance, public information campaigns to guide consumption, are fully and directly attributable to drug resistance. Other costs such as those associated with novel antibiotic R&D and antibiotic stewardship, infection control programs and associated training and implementation and monitoring costs, are also attributable to resistance. However, for most other components, especially at the individual level, attribution of cost to resistance is less clear. Methods to disentangle causation include subjective, labour-intensive chart review, or objective, costly cohort studies. In cohort studies, outcomes deriving from comparable patient groups with and without the drug-resistant infection/colonisation of interest are contrasted in order to measure the average additional cost imposed by the resistant form of the infection. Whether patients in the control groups should have a drug-susceptible infection, or should have no infection, is a subject of ongoing debate. Certain infections only happen because drug-resistance is present, like sepsis after an inappropriately treated urinary tract infection, or a surgical site infection after standard prophylaxis. In this case, a selection of control patients without infection is valid. For other infections this is much less clear. In certain cases the drug-resistant infection may just replace a drug-susceptible infection, and a selection of control patients with a drug-susceptible infection is most valid.AMR costing studies will likely need to derive estimates of attributable costs from cohort studies such as those described above. Unfortunately, not many, high quality studies exist, and often their external validity is limited, leaving little precedent to utilize as a guide.Furthermore, factors such as clinical manifestations – the distinction between colonisation and infection -- can add further layers of complexity when it comes to costs. For example, if the vague colonisation status of the patient is known, this is likely to increase costs: prophylaxis in the case of surgery or transplantation procedures would require a highly effective broader spectrum agent, which in many cases come with a higher price tag. Colonisation is also likely to lead to additional diagnostic tests, isolation of the patient, change in contact precautions (from standard precautions to standard plus contact precautions), and other costs. Conversely, while not knowing the colonisation status may lead to lower assumed costs, the costs associated with a transition to unanticipated resistant infection might be greater. The scenarios chosen to capture the different possible health states and their respective probabilities should reflect care realities at the local level .The transmission of resistance within and between communities today increases the risk of being colonized and therefore further reduce the therapeutic options for future patients.Finally, it should be noted that to comprehensively account for indirect costs may be particularly challenging, as highlighted in a recent framework developed to estimate the added value of new antibiotics in human health .For the purposes of this initial framework the starting point for selecting pathogen-drug pairs was the WHO list of priority pathogens for which new research is most urgently needed . This waAlso, while the choice was made to focus this framework largely on bacterial microbes in the first instance, following the WHO Global Action Plan , it shouFinally, although a species perspective was taken in this study, it is acknowledged that the microbiome, particularly those in the environment, include viable but not culturable (VNBC) bacteria that may also be reservoirs of resistance and resistance genes.Plasmodium falciparum, or HIV,) – none of which are explicitly listed in this work. In veterinary medicine anthelmintic resistance is an enormous problem already, whereas antibacterial resistance is probably not yet impacting on treatment of animal pathogens to the same extent as in humans. Resistance to antifungals also impose a non-negligible cost on healthcare services [Candida auris infection and the azole-resistant Aspergillus fumigatus [In focusing on bacteria, this work ignores costs imposed by resistance within other microorganisms. Even with regard to bacteria, the list is not exhaustive, as only major causes of disease/transmission/costs, were included. In some parts of the world the most worrying AMR will be amongst fungi, parasites, or viruses (e.g. services . Recent umigatus , 26. We Finally, in trying to create a framework that can be used by researchers or government worldwide to estimate the cost of AMR, this work is likely to miss some important details in how and where AMR imposes costs locally. Local studies may be needed to adapt the framework to clinical norms and the epidemiological reality to effectively capture costs.A further challenge is the lack of research capacity and funding, especially - but not only - in LMICs; the quality and accuracy of the data necessary to obtain reliable cost estimates is unlikely to be readily available in most countries.Attaining a realistic understanding of how and to what extent antibiotic resistance affects society is a challenging task. We hope that this work helps to pave the way to a clearer view of AMR costs and ultimately helps inform important decisions across the interconnected domains of human, animal, and environmental health in the years to come. Whether these decisions concern potential infection control interventions, targeting of surveillance efforts, how best to steer research and development efforts, or exciting innovative new ways of tackling AMR, a credible and nuanced assessment of AMR-related costs is essential. Using a sufficiently granular, bottom-up framework across multiple sites we should be able to achieve the necessary global estimates needed to support major international initiatives and better guide major R&D funding, while remaining sufficiently flexible to adapt to local realities and guide resource allocation.


Culex mosquito plays a central role in its transmission cycle. Culex mosquitoes harbor a wide range of viruses, including insect-specific viruses (ISVs), and can transmit a variety of arthropod-borne viruses (arboviruses) that cause human and animal diseases. The current trend of studies displays enhanced efforts to characterize the mosquito virome through bulk RNA sequencing due to possible arbovirus–ISV interactions; however, the extent of viral diversity in the mosquito taxon is still poorly understood, particularly in some disease vectors. In this study, arboviral screening and RNA virome analysis of Culex tritaeniorhynchus and C. pseudovishnui, which are part of the Culex vishnui subgroup mosquitoes, were performed. Results from these two mosquito species, known as the major vectors of JE virus (JEV) in Asia, collected in three prefectures in Japan were also compared with the sympatric species C. inatomii. A total of 27 viruses, including JEV, were detected from these Culex mosquitoes. Molecular and phylogenetic analyses of the detected viruses classified 15 of the 27 viruses as novel species, notably belonging to the Flaviviridae, Rhabdoviridae, Totiviridae, and Iflaviridae families. The successful isolation of JEV genotype I confirmed its continuous presence in Japan, suggesting the need for periodic surveillance. Aside from JEV, this study has also reported the diversity of the RNA virome of disease vectors and broadened the knowledge on mosquito virome profiles containing both arbovirus and ISV. Mosquito taxon seemed to contribute largely to the virome structure  as opposed to the geographical location of the mosquito species. This study therefore offers notable insights into the ecology and evolution of each identified virus and viral family. To the authors’ knowledge, this is the first study to characterize the viromes of the major JE vectors in Japan.Japanese encephalitis (JE) remains a public health concern in several countries, and the  Culex vishnui subgroup  viruses , and negative-sense ssRNA [(−)ssRNA] viruses . Further details of each virus are described below.The metagenome sequencing method uncovered a wide range of viruses from a total of 652 mosquitoes grouped into 13 pools . In totaCulex vishnui subgroup mosquitoes have the exact same totivirus beyond collection sites , whereas C. inatomii mosquitoes have a different totivirus   A. CvsTV up pools . CvsTV pup pools B, whereaC. tritaeniorhynchus originating from Ishikawa . Culex pseudovishnui has a different novel partitivirus named Culex pseudovishnui partitivirus (CpPV). CtPV formed a cluster with Hubei partiti-like virus 19, Hubei partiti-like virus 18, and Galbut virus, whereas CpPV displayed a correlation with Hubei partiti-like virus 56 and Changjiang partiti-like virus 1 ssRNA viruses. As many as 15 (+)ssRNA viral genomes were identified. Some were previously described as ISVs, some were novels, and one was a mosquito-borne arbovirus.Culex tritaeniorhynchus flavi-like virus (CtFLV), and JEV. A strain of mosquito flavivirus, described as Yamadai flavivirus, was found in two C. pseudovishnui pools. CtFLV belonged to an unclassified flavivirus clade together with viruses discovered from arthropods: Gamboa mosquito virus, Macrosiphum euphorbiae virus 1, Shayang fly virus 4, and Shuangao lacewing virus 2 . Another novel virus was identified from C. pseudovishnui and designated as Culex pseudovishnui negev-like virus (CpNLV). Phylogenetic analysis revealed that CtNLV formed a cluster with Mill Lade virus and Aedes camptorhynchus negev-like, whereas CpNLV formed a cluster with Yongsan negev-like virus (Negevirus was recently described as a diverse group of ISVs isolated from mosquitoes and phlebotomine sandflies . In thiske virus .C. pseudovishnui and designated as Culex pseudovishnui tymo-like virus (CpTLV). CpTLV formed a cluster with two recently identified viruses: Tarnsjo virus and Guadeloupe Culex-tymo-like virus ssRNA viruses, which currently includes five families. Most members of the order infect plants, but a few species are from plant pathogenic fungi . Generalke virus .C. tritaeniorhynchus and/or C. pseudovishnui: Wenzhou tombus-like virus 11 (WTLV11), Hubei mosquito virus 2 (HMV2), and Hubei mosquito virus 4 (HMV4) (C. inatomii luteo-like virus (CiLLV) was identified in C. inatomii. Based on the RdRp region, CiLLV was closely related to HMV2  . A novel to HMV2 ; likewis to HMV2 .C. vishnui subgroup mosquitoes but not in the C. inatomii library.For sobemoviruses, plants are their sole natural host. However, bulk RNA sequencing has increased the scope of their host range, with some viruses that belong to this group found in vertebrates and arthropods . In thisWSLV3 was reported to have 2841 bp nucleotides , shorterC. tritaeniorhynchus and C. pseudovishnui collected in Nagasaki; another strain of Yongsan iflavirus; and a novel iflavirus designated Yonago Culex iflavirus (YCIFV), which was detected from C. tritaeniorhynchus pools from Tottori. The phylogenetic tree displayed ICIFV as being in the same cluster with Bee iflavirus 1 and Wuhan insect virus 13, whereas YCIFV formed a cluster together with Kinkell virus and Wuhan fly virus 4 China (Iflaviruses are widely known as a group of picorna-like viruses that infect arthropods . In this 4 China .Five putative (−)ssRNA viruses, including major taxonomic categories, were discovered. Among these, one virus is a known mosquito virus, whereas the remaining four viruses are newly described in this study.Anphevirus [C. tritaeniorhynchus anphevirus (CtAV), was found in most pools of C. tritaeniorhynchus. Phylogenetic analysis showed a close relationship of CtAV with Guadeloupe mosquito mononega-like virus and Culex mononega-like virus 2  was solely identified in C. pseudovishnui. CpRLV displayed a close relationship with Tongilchon virus (Rhabdoviruses are a diverse set of (−)ssRNA viruses known to infect both animals and plants , with fron virus .C. pseudovishnui pools. The new bunya-like virus was tentatively named C. pseudovishnui bunya-like virus (CpBLV). The phylogenetic tree based on the RdRp sequences encoded by the L segment revealed CpBLV as being in the same clade as RdRp of the Bunyaviridae environmental sample and Culex bunyavirus 2, which possibly belong to the Phenuiviridae family ssRNA viruses, which currently includes more than 10 families ,41. In te family . Phylogee family Culex tritaeniorhynchus aspi-like virus (CtALV). CtALV was detected in a C. tritaeniorhynchus pool collected in Tottori. The phylogenetic tree showed that CtALV is closely related to Wilkie ophio-like virus 1 detected from A. camptorhynchus mosquitoes in Australia [In this study, a virus related to the Aspiviridae  family, was identified, which was designated ustralia  and ophiustralia  , and a novel iflavirus called YCIFV.C6/36 cells derived from  viruses  and to dAlthough the cell line used was mosquitoes, the isolated viruses were not limited to ISVs. Moreover, the fact that not every ISV was isolatable illustrated the cell line had variable sensitivity to virus infection. Lineages of the C6/36 cell line have been widely used to study the relationship between arboviruses and mosquito vectors  and theyC. tritaeniorhynchus. The fact that those undetected viruses belong to the Flavivirus genus, which contains many medically important viruses, highlighted the sense that, in arbovirus surveillance, the method of virus amplification through mosquito cell lines should not be overlooked.Three blind passages of virus isolation in mosquito cell lines amplified two viruses not detected by NGS: CxFV and mosquito flavivirus in the Nagasaki group’s Using the NGS result as the lead, primers were designed and RNA viruses in pools were confirmed by RT-PCR; thus, the virome of each mosquito species and collection sites can be analyzed and putative seasonal changes of virome composition can also be portrayed . There wC. tritaeniorhynchus belonging to the vishnui subgroup is the main vector in Japan [It is estimated that approximately 67,900 JE cases typically occur annually in 24 JE-endemic countries , includiin Japan . This stCulex mosquito species, C. tritaeniorhynchus and C. pseudovishnui renowned as important vectors for JEV in Asia and C. inatomii as the non-vector species, was analyzed to better understand arbovirus–ISV interaction. The abundance and diversity of possible ISVs constituted a significant component of Culex mosquito viromes; for example, the virome of Culex spp. in Zambezi, Mozambique was dominated (88.5%) by them [C. tritaeniorhynchus virome in Hubei, China also showed a conformable predominance (88%) of insect viruses [C. tritaeniorhynchus, C. pseudovishnui, and C. inatomii, respectively , whereas another Culex belonging to a different subgenus, C. inatomii, shared only one virus with mosquitoes collected in the same area. This finding corresponds to a study comparing viral diversity in some species of mosquitoes, which showed correlation between mosquito species and their virome structures [Metagenomic sequencing results suggested a distinct virus distribution depending on the mosquito taxonomy. Here, it was shown that two mosquitoes belonging to the same subgroup, ructures .C. tritaeniorhynchus, originated from various collection sites, and time has corroborated the taxon impact on virome shaping even more. The absence of these predominant viruses in other species could imply disparity in host susceptibility, although this would need further clarification.Recurring detection of certain predominant viruses from one mosquito species, Culex mosquito species inhabiting the same area. This phenomenon was vividly demonstrated by detection of HMV4, which was detected from the two mosquito species (C. tritaeniorhynchus and C. inatomii) collected from the same sites in Tottori. In some way, particular viruses present in this study were also discovered in geographically distant localities, such as HMV2 and WSLV3, which were already detected in C. tritaeniorhynchus in China [Culex sp. mosquitoes has long been speculated, before nucleotide identities (99% in both viruses) remarked an untypical limited genetic transformation despite the geographical distance. Again, this correlated with a previous finding that some viruses infected hosts over a wide geographical area and were possibly introduced by windblown, cyclones, or humans rather than the spread of mosquitoes across continents over time [Not only does this study display a pattern of host taxon as one of the virome-shaping factors, but it also reveals another point—that some viruses could be detected in discrete in China . In the ver time ,50,51,52C. tritaeniorhynchus from Ishikawa Prefecture. No other virus was considered as arbovirus based on their closest related viruses or phylogenetic tree. An interesting aspect exhibited from virus isolation might be that another flavivirus, CxFV, was not detected in the mosquito homogenate before virus isolation. Nagasaki Prefecture, where CxFV isolates were collected, is a reputable JE-endemic area in Japan, as JEV detection is frequent in annual disease surveillance of amplifiers and vectors. JEV was not isolated nor identified in 2017 Nagasaki samples, thus making it difficult to decipher the interrelation between the flaviviruses. Although an in vitro study by Kuwata et al. [C. tritaeniorhynchus mosquitoes, this study was unable to demonstrate the correlation between CxFV occurrence and the detection or otherwise of JEV.Aside from the patterns observed, this study also discusses the viral association with hosts and possible interrelation with other coexisting viruses. The arbovirus transmitted by mosquitoes detected in the study was JEV, which was identified in a et al.  implied The only collection site where JEV was identified is Monzenmachi-toge, Ishikawa Prefecture, where the landscape is a vast rice field surrounded by forest with the river and sea in close proximity. The result was supported by annual disease surveillance conducted in September 2017 by the Japan National Institute of Infectious Disease (2017) , which hAlphavirus, family Togaviridae). A study reported the virus being transported in the same direction from west Japan into central Japan, with possible routes of transportation, such as bird migration and windblown mosquitoes [Phylogenetic analysis of the complete genome showed that the isolated JEV strain 17CxIT-I4-D31 was closely related to JEV isolates detected in Yamaguchi in 2013 rather than those from Ishikawa previously or other surrounding areas, suggesting that JEV may be transported from west Japan into central Japan. The phenomenon of this virus spread has also occurred in Getah virus  was first identified from pooled organs  of  in 2009 . Yet, ite virome ,58. Howee virome ; additioe virome ,61. TherNevertheless, the bat sobemovirus nucleotide sequence identified by Dacheux et al. (2014)  was a viImportantly, this mosquito virome study was carried out using metagenomic sequencing in combination with mosquito cell line virus isolation. Efforts have been put into metagenomic analysis using NGS for disease surveillance, as the method allows the simultaneous identification of viruses from a single mosquito in a single reaction . HoweverThe combination of high-throughput sequencing and virus isolation in this study was meant to retrieve the complete mosquito virome, including underlying viruses that exist at very low copy numbers, whereupon the results can be interpreted. C6/36 cells were selected, as they are easy to maintain and highly permissive to numerous arboviruses , althougCulex mosquito virome study in Japan. In addition to JEV and several known ISVs, other novel viral sequences were identified from these mosquitoes. Notwithstanding, further studies will be required to confirm the real host of the identified viruses as the metatranscriptomic studies may also include non-mosquito RNA. By studying one of the medically important disease vectors, this study offers important insights into the ecology and evolution of mosquito viromes consisting of both arbovirus and insect viruses.Finally, complete analysis was performed on the virome profile of major JE vectors in Japan, including an arbovirus, ISVs, and other viruses. This is the first report on the


Grapevine viruses are found throughout the viticultural world and have detrimental effects on vine productivity and grape and wine quality. This report provides a comprehensive and up-to-date review on grapevine viruses in Australia with a focus on “Shiraz Disease” (SD) and its two major associated viruses, grapevine virus A (GVA) and grapevine leafroll-associated virus 3 (GLRaV-3). Sensitive grapevine cultivars like Shiraz infected with GVA alone or with a co-infection of a leafroll virus, primarily GLRaV-3, show symptoms of SD leading to significant yield and quality reductions in Australia and in South Africa. Symptom descriptors for SD will be outlined and a phylogenetic tree will be presented indicating the SD-associated isolates of GVA in both countries belong to the same clade. Virus transmission, which occurs through infected propagation material, grafting, and naturally vectored by mealybugs and scale insects, will be discussed. Laboratory and field-based indexing will also be discussed along with management strategies including rogueing and replanting certified stock that decrease the incidence and spread of SD. Finally, we present several cases of SD incidence in South Australian vineyards and their effects on vine productivity. We conclude by offering strategies for virus detection and management that can be adopted by viticulturists. Novel technologies such as high throughput sequencing and remote sensing for virus detection will be outlined. Vitis vinifera, cv. Shiraz (syn. Syrah) is the most popular cultivar in Australia. In 2018, of the total of 135,133 ha under cultivation, Shiraz accounted for 39,893 ha  making it the most widely planted winegrape cultivar in Australia . Of 86 viruses detected in the grapevine to date  V. vinifera cv. Shiraz (syn. Syrah), symptoms of primary bud necrosis (PBN) are often observed in buds during the dormant season  typically appear around véraison. However, the virus can be detected using molecular techniques at the pre-véraison stage . Basal lRed grape cultivars are more sensitive to GLD and can develop dark red or purple colouration in the interveinal sections of the leaf, often with distinct green veins b, while Although some leafroll viruses may produce SD-like symptoms , GVA hasSeveral full-length and partial sequences of SD associated viruses obtained in this study using HTS are listed in The sequence of the coat protein gene of the Australian GVA isolates was compared with 40 other isolates available on GenBank, and a phylogenetic tree was constructed . The treFor GLRaV-3, the phylogenetic study was based on nearly complete sequence of the viral CP gene . A totalIt is interesting to note that in R3ShRam andR4ShRam that were isolated from the same Shiraz vineyard in Riverland, SA, two different groups of GLRaV-3 (Groups I and IV) were detected . Table 5Two mild isolates of GLRaV-3 were detected in Australia, both belonging to Group VII. CSL isolated from Crimson Seedless (Accession MT081182) table grapes from Western Australia showed mild leafroll symptoms; this isolate clustered with a symptomless isolate 139  of the va fluorescence, processes that directly or indirectly relate to the vine’s ability to maintain vegetative and reproductive growth as well as ripen crop. Both photosynthesis and chlorophyll fluorescence were observed to be lower in GLRaV-3-infected Cabernet Franc grapevines in a Michigan (USA) vineyard compared to healthy vines after véraison [Virus-related diseases can affect grapevine physiological performance, vigour, yield, grape and wine composition, and quality ,75. Chanvéraison . In the véraison . Yield lvéraison ,79,80,81véraison . In extrA study on Cabernet Sauvignon grapevines in Chile found that the main anthocyanin and sugar metabolism genes were down-regulated during fruit ripening in GLRaV-3-infected vines . In PinoA preliminary study in Southern Oregon, USA, on Grapevine red blotch virus (GRBV)-infected Cabernet Franc grapevines indicated that virus infections can be detected prior to véraison before the appearance of red leaf symptoms based on measurements of leaf chlorophyll fluorescence . Leaf reddening may be a response of grapevines to virus infection in which they accumulate anthocyanins that play a protective role as antioxidants in scavenging free radicals produced by the vine under biotic stress . Nearly Conversely, some virus species and isolates may not cause disease or impact yield or grape quality particularly when they infect grapevines in the absence of other viruses . InteresFuture studies could also consider investigating virus effects on grape berry development and potentially delayed ripening, which are becoming increasingly important in the context of climate change and regional warming ,90.Both GLD and SD are debilitating to vineyards due to economic losses associated with yield reductions, lower grape prices due to inferior fruit quality , and vin−1 yr−1 for prevention and control [−1. Some conscious growers, or those whose economic impact is more significant, opt to rogue and replant the vines and even entire vineyards thereby decreasing the risk of virus spreading to neighbouring vines and vineyards. In Australia, the lifespan of a vineyard infected with SD may not exceed six years and is typically destroyed as it becomes economically unviable. In 2012 in the McLaren Vale and Barossa Valley viticulture regions of South Australia, entire blocks of Shiraz vines grafted on to GVA positive Chardonnay were removed six years after grafting due to lack of productivity . It is estimated that the cost of removing virus infected vines and replanting in Australia is around AU$70,000 ha−1 [In Australia, many grape growers may be complacent in their management of virus infections as they consider the magnitude of the issue insignificant relative to other pathogens in the vineyard including trunk diseases  and powdery mildew. As a result, virus-infected vines are often ignored. A report on the economic impact of virus and related-infections on grapevines indicated that the reduced profit ranged from AU$34 to $103 ha control . In the both visual symptoms and confirmation with molecular diagnosis; and (iii) replanting with certified plant material from an established grapevine nursery with clean source blocks and good sanitation practices.Virus-infected grapevines serve as inoculum for vector-based spreading of viruses in vineyards. Inaction will likely result in the spread of viruses within and between vineyard blocks resulting in larger pools of inoculum, much like fungal diseases that are better known to viticulturists. Atallah and co-workers modelled the spread of disease within and between blocks and found that costs of managing the disease are overestimated when only inter-block spatial dynamics, e.g., virus transmission from neighbouring vineyards via vectors, compared to when the effects of within-block disease spread are additionally considered . SeveralErysiphe necator) may indirectly contribute to increases in virus vector populations via their negative effects on parasitoids [Routine scouting of vineyard blocks for both virus symptoms and potential vectors such as mealybugs and scale insects should be done to minimize disruption to vine productivity and reduce economic losses associated with virus-related diseases. A combination of systemic and contact insecticides to control mealybugs and scales, in particular at the early stage in the lifecycle, i.e., first and second instar nymphs, or the use of biological control agents, e.g., parasitoids and mating disruption (pheromone traps), can be effective in minimizing the spread of GVA and GLRaV-3 in vineyards . The useasitoids .Parthenolecanium persicae), which are the predominant scale species in Australian vineyards [GVA and GLRaV-3 have been detected in and transmitted by, amongst others, grapevine scale insects . These symptoms vary based on phenological stage, environmental conditions, as well as cultivar. In this section, we provide specific regional examples of SD and the symptoms elicited.Over several seasons, observations of mature (established) grapevines indicated consistent patterns of SD expression in regional vineyards across South Australia, the state with the largest winegrape area in Australia . The cultivar specificity of SD was observed in the same vineyard: adjacent rows of Chardonnay grapevines tested positive for GVA and occasionally for GLRaV-3 but did not show vine decline  shoot growth during early stages of vine development. We observed retarded shoot growth symptoms in vineyards in Riverland, McLaren Vale, and Langhorne Creek regions b,c. ShooIn summary, our observations of SD in Australian vineyards indicate that the disease has the potential to cause vine decline manifested in delayed or no bud burst, retarded shoot development, and low or no crop. SD appears to be cultivar specific, with Shiraz and Malbec showing typical SD symptoms. Other red-berried cultivars such as Gamay, Merlot and Sumoll are also known to show SD symptoms, while Cabernet Sauvignon does not despite specific clones, e.g., SA125 and Reynella, that are infected with GVA.Bunyavirales [GVA and GLRaV-3 pose an ongoing threat to Australian vineyards due to their negative consequences on vine vigour, yield, and economic returns to the grower. In a few extreme cases, vineyards experienced yield losses of up to 98%, underscoring the importance of improving our understanding of these viruses; their source; methods of spread; potential vectors and their control; differences in cultivar susceptibility; and, most importantly, the specific interaction between the virus species on grapevine productivity. Systematic research needs to be undertaken to shed light on these open questions and to determine the long-term implications of virus-infected blocks including developing the best strategies to maintain their viability. Advances in proximal and remote sensing technologies provide opportunities for non-destructive detection of virus-infected grapevines ,107 in aavirales , should