Mutated COVID-19, May Foretells

Mankind in a Great Risk in the Future

Ali A. Dawood
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Mutated COVID-19, May Foretells Mankind in a Great Risk in the Future

Ali A. Dawood, Ph.D

PII: S2052-2975(20)30025-1
DOI: https://doi.org/10.1016/j.nmni.2020.100673
Reference: NMNI 100673

To appear in: New Microbes and New Infections

Received Date: 27 March 2020

Accepted Date: 30 March 2020

Please cite this article as: Dawood AA, Mutated COVID-19, May Foretells Mankind in a Great Risk in the
Future, New Microbes and New Infections, https://doi.org/10.1016/j.nmni.2020.100673.

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© 2020 Published by Elsevier Ltd.

Mutated COVID-19, May Foretells Mankind in a Great Risk in The
Future
Ali A. Dawood
Ph.D. Microbiology, College of Medicine, University of Mosul, Mosul, Iraq

Abstract:
Corona virus disease 2019 SARS-CoV-2 (COVID-19) is a zoonotic virus causing a variety of
severe of respiratory diseases. SARS-CoV-2 is closest to SARS-CoV and MERS-CoV in
structure. The highly prevalence of COVID-19 is due to the lack onset of symptoms. Our study
aimed to present an overview of the virus in terms of structure, epidemiology, symptoms,
treatment, and prevention. Conduct the differences of whole genome sequence and some viral
proteins to determine the gap and the change alternation of nucleotides and amino acids
sequences. We evaluate 11 complete genome sequence of different coronavirus using BAST and
MAFFT software. We also selected 7 types of structural proteins. We were conclude that
COVID-19 might be created new mutations specifically in glycoproteins hence requires caution
and complete preparation by health authorities.
Keywords: COVID-19, MERS, outbreaks, SARS, zoonotic.
ORCID: 0000-0001-8988-5957, Email: aliadeldawood@gmail.com

Introduction:
The first emerging of novel coronavirus disease 19 was 31th, December 2019 in Wuhan city,
China. COVID-19 is classified the seventh member of the subfamily Orthocoronavirinae under
the family Coronaviridae. The most members of this family are zoonotic viruses that transmitted
to humans via contact with infected animals. Although the bats and snakes are the natural
reservoir of wide coronaviruses, there is no evidence so far that the COVID-19 was originated
and transmitted from the seafood market [1]. Previous study reported that lipid rafts of
coronaviruses have made new strain COVID-19 which is identity 80% to SARS-CoV. Lipid
molecules such as caveolins, clathrins and dynamin have a fundamental role in the
internalization of viruses. Firstly, these molecules are involved in the entry of viruses into host
cells. Second, targeting host lipids is being studied as an antiviral strategy and could have
various applications [2]. COVID-19 seems to need to bind to the ACE-2 receptor on the
membrane host cell to enable it to infect host cell upon coupled with a reliance of serine protease
TMPRSS2. This intracellular protein seems to be a determinant of the virus ability to infect cell
[3].
Mutated COVID-19, May Foretells Mankind in a Great Risk in The
Future

Abstract:
Corona virus disease 2019 SARS-CoV-2 (COVID-19) is a zoonotic virus causing a variety of
severe of respiratory diseases. SARS-CoV-2 is closest to SARS-CoV and MERS-CoV in
structure. The highly prevalence of COVID-19 is due to the lack onset of symptoms. Our study
aimed to present an overview of the virus in terms of structure, epidemiology, symptoms,
treatment, and prevention. Conduct the differences of whole genome sequence and some viral
proteins to determine the gap and the change alternation of nucleotides and amino acids
sequences. We evaluate 11 complete genome sequence of different coronavirus using BAST and
MAFFT software. We also selected 7 types of structural proteins. We were conclude that
COVID-19 might be created new mutations specifically in glycoproteins hence requires caution
and complete preparation by health authorities.
Keywords: COVID-19, MERS, outbreaks, SARS, zoonotic.

Introduction:
The first emerging of novel coronavirus disease 19 was 31th, December 2019 in Wuhan city,
China. COVID-19 is classified the seventh member of the subfamily Orthocoronavirinae under
the family Coronaviridae. The most members of this family are zoonotic viruses that transmitted
to humans via contact with infected animals. Although the bats and snakes are the natural
reservoir of wide coronaviruses, there is no evidence so far that the COVID-19 was originated
and transmitted from the seafood market [1]. Previous study reported that lipid rafts of
coronaviruses have made new strain COVID-19 which is identity 80% to SARS-CoV. Lipid
molecules such as caveolins, clathrins and dynamin have a fundamental role in the
internalization of viruses. Firstly, these molecules are involved in the entry of viruses into host
cells. Second, targeting host lipids is being studied as an antiviral strategy and could have
various applications [2]. COVID-19 seems to need to bind to the ACE-2 receptor on the
membrane host cell to enable it to infect host cell upon coupled with a reliance of serine protease
TMPRSS2. This intracellular protein seems to be a determinant of the virus ability to infect cell
[3].
Prevalence and epidemiology:
Over the past two decades, outbreaks of coronavirus have been observed, the Severe Acute
Respiratory Syndrome (SARS) - COV in 2003 and Middle East Respiratory Syndrome (MERS)
– CoV previously described as a major public health threat. Nowadays, the World Health
Organization considers COVID-19 is more serious and widespread epidemic disease [4]. To
date, it seems that the mortality rate of COVID-19 is lower than the incidence of SARS or
MERS. A significant increase number of COVID-19 cases was observed due to the absence of
emerging pathological symptoms in the virus carriers. For this reason, it may foster the collapse
of local health care [4]. Some countries face an outbreak crisis and try to prevent the spread of
COVID-19 through preventing human gatherings, a curfew is imposed in cities, prevent travel
between countries and close the land borders may reduce the outbreaks.
The main transmission of COVID-19 starts with contacted human to human including relatives
and friends who intimately contacted with patients or incubation carriers. Many studies reported
that coughing and sneezing are the quicker way of the virus dispersion as well as droplet, and
airborne precautions when encountering an infected person [5].
Virus structure:
COVID-19 relates to the betacoronavirus that infects humans and likely developed from bat
origin coronaviruses. Structural analysis shows that COVID-19 probably derives from a bat
SARS-like coronavirus, which has mutated in the spike glycoprotein (protein S) and
nucleocapsid N protein The positive-sense RNA genomes of COVID-19 is differ from SARS-
CoV and MERS-CoV approximately 29.9 kb, 27.9 kb and 30.1 kb, respectively [6]. The
COVID-19 complete genome was annotated to possess 14 open reading frames ORFs encode 27
proteins. Sequence analysis revealed that COVID-19 is identity 80% more than to SARS-CoV
and 50% to the MERS-CoV which originated in bat [7, 8]. In addition to that, spherical external
spike protein displays a characteristic crown shape can be observed under an electron
microscope [9]. Current study, we have compared between novel COVID-19 complete genome
with other related corona virus to provoke the mutation and the gaps. We selected the data from
NCBI and we did the FASTA and BLAST. The comparison between genomes with alignment
has done using MAAFT-7 software. COVID-19 gene bank (MT188341.1), COVID-19
(MT066175.1), bat-SL-CoVZC45 (MG772933.1), SARS-CoV BJ182b (EU371561.1) are
identical alignment in 99%, 89%, and 82% respectively. In figure (1) shows the differences
between 4 complete genomes shows as follow:
GGTATGAGCTATTATTGTAAATCACATAAACCGCCCATTAGTTTTCCATTGTGTGCTAAT 16440
................................A........................... 16494

GGACTACCAACTCAAACTGTTGATTCATCACAGGGCTCAGAATGTGACTATGTCATATTC 17820
...........................................A................ 17874

GGACTTTTTAAAGATTGTAGTAAGGTAATCACTGGGTTACATCCTACACAGGCACCTACA 18060
.....C...................................................... 18114

MT188341.1 + MT066175.1 similarity 99% partial seq.

TCATCAAACGTTCGGATGCTCGAACTGCACCTCATGGTCATGTTATGGTTGAGCTGGTAG 479
.............T.....C..............C..C...........C..AT.A.... 532

CAGAACTCGAAGGCATTCAGTACGGTCGTAGTGGTGAGACACTTGGTGTCCTTGTCCCTC 539
..........T...........T..........................T.......... 592

ATGTGGGCGAAATACCAGTGGCTTACCGCAAGGTTCTTCTTCGTAAGAACGGTAATAAAG
....A..A..GG.......T........T..A............................ 652
MT188341.1 & MG772933.1 similarity 89% partial seq.
GAAAACTTGTTACT-TTATAT--TGACATTAATGGCAATCTTCATCCA-GATTCTGCCAC 4017
A.T..---......C..G.T.GC...T..C.....T..G...T.-...T......-.AGA 3997

TCTTG-TT--A-GTGACATTGACATCACTTTCTTAAAGAAAGATGCTCCATATATAGTGG 4073
A.A..C..AG.G....-.--..T..GT.....C.TG....G.....A..T..C..G..A. 4054

GTGATGTTGTTCAAGAG-GGTGTTTTAACTGCTGTGGTTATACCTACTAAAAAGGCTGGT 4132
........-A...CT..T....A.A.C...TG...T..A.....CT.C............ 4113

MT188341.1 & EU371561.1 similarity 82% partial seq.

Figure (1): Final alignment between 4 complete genome

According to the alignment analysis, there is a closest similarity between 2 COVID-19 in 99%
compared with other 2 Bat CoV. 7 complete genomes have been alignment for different
coronavirus stains figure (2).

MT066175.1 SARS-CoV-2/NTU01/2020/TWN
MT039873.1 SARS-CoV-2 HZ-1

MN997409.1 SARS-CoV-2/USA-AZ1/2020

MN996532.1 Bat CoV RaTG13

EU371564.1 SARS-CoV -2 BJ182-12

KY938558.1 Bat CoV 16BO133

MN985325.1 SARS-CoV-2/USA-WA1/2020

Figure (2): MAFFT considers similarities in forward strands (red) only, but ignores similarities in reverse strands
(blue), Plot 1/2 alignment MT066175.1 + MT039873.1= 99.7%, plot 1/3: MT066175.1+ MN997409.1=99.7%, plot
1/4 MT066175.1+ MN996532.1= 89%, plot 1/5 MT066175.1+ EU371564.1= 82.4%, plot 1/6 MT066175.1+
KY938558.1= 68%, plot MT066175.1+ MN985325.1= 95%.

Upon to the result above, we revealed that all COVID-19 strains are closest similarity compared
to other strains related to the same family. In other words, we believe that COVID-19 came from
several mutations happened to other members of coronavirus relates to the same infection.
Although genomic analysis does not support the belief that COVID-19 is a laboratory construct,
currently it is impossible to disprove or prove the theories of its origin. To identify the COVID-
19 origin, obtaining virus sequences from immediate animal sources would be the most definite
method.
The first ORF (ORF1a/b) translates two polyproteins, pp1a and pp1ab, and encodes 16 non-
structural proteins (NSP) which takes two-thirds of viral RNA. The remaining ORFs encode
structural proteins including spike (S) glycoprotein, small envelope (E) protein, matrix (M)
protein, and nucleocapsid (N) protein. COVID-19 also possesses accessory proteins that interfere
with the host innate immune response [8].
We have done alignment for different strain of ORF10 COVID-19 GenBank: (QIK50446.1) with
the ORF10 COVID-19 (YP_009725255.1). The result gave similarity 100% without any
mutation in amino acid figure (3).
MGYINVFAFPFTIYSLLLCRMNSRNYIAQVDVVNFNLT 38

...................................... 38

Figure (3): 100% similarity between ORF 10 (QIK50446.1) and (YP_009725255.1).

Nucleocapsid phosphoprotein COVID-19 GenBank: (QIK50445.1) has been aligned with NP

COVID-19 (QIC50514.1) similarity 100%, NP SARS CoV CUHK-L2 (AAS01074.1) similarity
85%, and bat COV HKU5 (QHA24694.1) similarity 61% figure (4). Crystal structure of NP
COVID-19 (6VYO) has been released in 11/3/2020 from RCSB PDB figure (5).
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHG 60
............................................................ 60
KEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAG 120
............................................................ 120
(QIK50445.1) + (QIC50514.1) similarity 100%
GKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEA 119
...E.R.............G..............V........E................ 120
GLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGG 179
S.......E..V.....XXXXXXXXXXXXXXXXXXXXXXXX................... 180

(QIK50445.1) + (AAS01074.1) similarity 85%

RATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGT 148
.QD.K.NT.N.-T.P.A.......T......N..FRSV.......HEN..TDA.-SVF.. 136
RNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPA 208
.....DP...T.FAP......N.HI..TG.N..S...A....SSR.SSRNGR.NNSSRN. 196
(QIK50445.1) (QHA24694.1) similarity 61%

Figure (4): alignment between (QIK50445.1), (QIC50514.1), (AAS01074.1) and (QHA24694.1).

Figure (5): Crystal structure of NP COVID-19 (6VYO) from RCSB PDB.

ORF7a protein COVID-19 GenBank: (QIK50443.1) has been aligned with NS7a Bat CoV
RaTG13 (QHR63305.1) similarity 99%, hypothetical protein SARS 7 CoV (AFR58706.1)
similarity 89% and putative uncharacterized protein 4 SARS CoV (AAX16199.1) similarity 68%
figure (6).
MKIILFLALITLATCELYHYQECVRGTTVLLKEPCSSGTYEGNSPFHPLADNKFALTCFS 60
.......V.V.................................................. 60
TQFAFACPDGVKHVYQLRARSVSPKLFIRQEEVQELYSPIFLIVAAIVFITLCFTLKRKT 120
...........................................I................ 120
(QIK50443.1) + (QHR63305.1) similarity 99%

MKIILFLALITLATCELYHYQECVRGTTVLLKEPCSSGTYEGNSPFHPLADNKFALTCFS 60
.......T..VFTS.....................P......................T. 60
TQFAFACPDGVKHVYQLRARSVSPKLFIRQEEV-QELYSPIFLIVAAIVFITLCFTLKRK 119
.H.....A..TR.T.........X.........Q......L......L..LI....I... 120
(QIK50443.1) + (AFR58706.1) similarity 89%
MKIILFLALITLATCELYHYQECVRGTTVLLKEPCSSGTYEGNSPFH-PLADNKFALTCF 59
.......T..VFTS.....................P..........AI.CFN.AYYILV. 60
STQFAFACPDGVKHVYQ 76
TRN-----.GSRRTL.. 72
(QIK50443.1) + (AAX16199.1) similarity 68%

Figure (6): alignment between (QIK50443.1), (QHR63305.1), (AFR58706.1) and (AAX16199.1).

ORF8 protein COVID-19 GenBank: (QIK50444.1) has been alignment with ORF8 protein
COVID-19 (QHN73801.1) similarity 99%, hypothetical protein Bat SARS CoV Rs806/2006
(ACU31050.1) similarity 76% and hypothetical protein Bat SARS CoV HKU3-8 (ADE34775.1)
similarity 84% figure (7).
MKFLVFLGIITTVAAFHQECSLQSCTQHQPYVVDDPCPIHFYSKWYIRVGARKSAPLIEL 60
............................................................ 60
CVDEAGSKSPIQYIDIGNYTVSCLPFTINCQEPKLGSLVVRCSFYEDFLEYHDVRVVLDF 120
.......................S.................................... 120
(QIK50444.1) + (QHN73801.1) similarity 99%
MKFLVFLGIITTVAAFHQECSLQSCTQHQPYVVDDPCPIHFYSKWYIRVGARKSAPLIEL 60
..L.IVF.LL.P.YCI.K...I.E.CEN...QIE......Y..D.F.KI.S....R.VQ. 60
CVDEAGSKSPIQYIDIGNYTVSCLPFTINCQEPKLGSLVVRCSFYEDFLEYHDVRVVLDF 120
.EGDY.KRI..H.EMF....I..E.LE....A.PV...I....YDY..V.H......... 120
(QIK50444.1) + (ACU31050.1) similarity 76%

GNYTVSCLPFTINCQEPKLGSLVVRCSFYEDFLEYHDVRVVLDFI 121
....I..E.LE....A.PV...I....YDY..V.H.......... 47
(QIK50444.1) + (ADE34775.1) similarity 84%

Figure (7): alignment between (QIK50444.1), (QHN73801.1), (ACU31050.1) and (ADE34775.1).

ORF6 protein COVID-19 GenBank: (QIK50442.1) has been aligned with ORF6 protein COVID-
19 (QIG55989.1) similarity 98%, protein 7 Rhinolophus affinis CoV (AHX37562.1) similarity
88% and NSP 6 SARS CoV ExoN1 (AGT21083.1) similarity 86% figure (8).
MFHLVDFQVTIAEILLIIMRTFKVSIWNLDYIINLIIKNLSKSLTENKYSQLDEEQPMEI 60
.............................................V.............. 60
(QIK50442.1) + (QIG55989.1) similarity 98%

MFHLVDFQVTIAEILLIIMRTFKVSIWNLDYIINLIIKNLSKSLTENKYSQLDEEQPMEI 60
...............I.......IA.....V..SS.VRQ.F.P..KKN..E..D.E.... 60
(QIK50442.1) + (AHX37562.1) similarity 88%
MFHLVDFQVTIAEILLIIMRTFKVSIWNLDYIINLIIKNLSKSLTENKYSQLDEEQPMEI 60
..............SI......RIA.....V..SS.VRQ.L.P..KKN..E..D.E...L 60
(QIK50442.1) (AGT21083.1) similarity 86%

Figure (8): alignment between (QIK50442.1), (QIG55989.1), (AHX37562.1) and (AGT21083.1).

Membrane glycoprotein COVID-19 GenBank: (QIK50441.1) has been aligned with MG

COVID-19 (QIG55988.1) similarity 99%, M protein COVID-19 (APO40582.1) similarity 93%
and membrane glycoprotein Rousettus Bat CoV HKU9 (YP_001039974.1) similarity 61% figure
(9).
MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPV 60
........................................................V... 60
TLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILL 120
............................R............................... 120
(QIK50441.1) + (QIG55988.1) similarity 99%
SNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVTLA 63
E.D....DQ..H.............FA..L......S...........V.......I... 62
CFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILLNVP 123
............A....................V...........W.............. 122
(QIK50441.1) + (APO40582.1) similarity 93%
DSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVTL 62
NCTN.VPRP.VIAA.KD..FAVSVIL.FITV...WG.PS.CKPIWV..MFI......LSI 63
ACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILLNV 122
.AA.F..IHP..SVAF.F...F..IS.I.......S....LC..G.A...M...DM.I.I 123

(QIK50441.1) + (YP_001039974.1) similarity 61%

Figure (9): alignment between (QIK50441.1), (QIG55988.1), (APO40582.1) and (YP_001039974.1).

Envelope protein COVID-19 GenBank: (QIK50440.1) has been aligned with EP COVID-19
(QHZ00381.1) similarity 98%, Chain A, Envelope small membrane protein SARS CoV
(5X29_A) similarity 90% and envelope protein Hypsugo Bat CoV HKU25 (ASL68947.1)
similarity 56% figure (10).
MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYVYS 60
....................................H....................... 60
(QIK50440.1) + (QHZ00381.1) similarity 98%

FVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVK 63
.Q.M................................A..AA..........TV....... 79
(QIK50440.1) + (5X29_A) similarity 90%
MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYVYS 60
.LP..Q.QI.SF...FFIFTV.CAIT...CM.F...T...MQ.AIG..TL..Q.AI...N 60
(QIK50440.1 (ASL68947.1) similarity 56%

Figure (10): alignment between (QIK50440.1), (QHZ00381.1), (5X29_A) and (ASL68947.1).

By analyzing the series compatibility of the proteins sequences under study, we can confirm that
there is a match between strains of COVID-19. There are obvious differences with respect
compared to other species of coronavirus family. This may indicate that COVID-19 originated
from mutations happened in coronavirus family. In clearer terms, new mutations may be created
as there in a high probability, specifically in glycoproteins.
We are unable to give reasonable explanations for the significant number of amino acid
substitutions between the COVID-19 and SARS-CoV or MERS-CoV due to very limited
knowledge of this novel virus.
Clinical manifestation and symptoms:
The incubation period of the virus may dissimilar accordingly to the age and immune status. As a
general, it has been assumed that incubation period sites between 2-14 days while some cases
observed till 23 days after exposure. The main symptoms can easily seem with elderly aged
above 70 and immunocompromised and diabetic patients. The symptom starts with fever, dry
cough, dyspnea, as well as sore throat, nasal congestion, malaise and bilateral infiltrates may be
seen on chest X-ray, however some cases are detected absence of fever. Clinical features of
COVID-19 include the targeting of the lower airway as evident by upper respiratory tract
symptoms like rhinorrhoea, sneezing, and sore throat which is developed to gastrointestinal
symptoms like diarrhea [10]. Severe cases may present with sepsis, heart attack or even shock.
Conversely, some cases may show mildly ill or asymptomatic altogether.
From WHO records, the period from the symptoms onset and to death of COVID-19 ranged
from 6 - 41 days with a median of 14 days. This period depends on the age and the status of
immune system. It has been shorter with age under 70 years [11].
Preventions:
To prevent spreading virus, managed care of patients with entails early identification. Rapid
isolation, timely establishment of infection prevention and control measures, together with
symptomatic care for patients with mild disease. Supportive treatment for those with severe
COVID-19. Specific attention and spend more efforts to reduce transmission should be presented
to susceptible populations including health care providers, immunocompromised patients,
children, and elderly people [5]. Health care systems around the world must operate with more
than one maximum capacity. It is necessary to cooperate between HCS and WHO to reduce
infection. The use of international media, social media and societal culture by maintaining
personal cleaning, minimizing risk of exposure, avoiding gatherings and preventing all
phenomena that lead to contact between persons [12]. COVID-19 vaccines is under accelerated
development.
The global public health community have to consider the effects of mass gathering cancellations
on the future wellbeing of communities through economic recession as well as through the
spread, or otherwise, of COVID-19 [13].
Diagnosis:
Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is the most specific and
sensitive assay approved and straightforwardly used by many reference laboratories worldwide.
Other laboratory tests may help assessing disease severity and predicting the risk of evolution
such as cute respiratory distress syndrome (ARDS) disseminated intravascular coagulation (DIC)
and multiorgan failure (MOF). Moreover, C reactive protein (CRP), lactate dehydrogenase
(LDH), erythrocyte sedimentation rate (ESR) and D-dimer, along with diminished concentration
of serum albumin, increased values of LDH, aspartate aminotransferase (AST), alanine
aminotransferase (ALT), total bilirubin, creatinine, cardiac troponins, are used to enhanced and
helpful tests for organs function. Notably, a combined IgM-IgG rapid immunoassay has also
been recently developed as well as elevation of pro-inflammatory cytokines detection kits such
as IL1-β, IL1RA, IL7, IL8, IL9, IL10, basic GCSF, IFNγ, TNFα,and IP10 [14]. A study revealed
that gastrointestinal symptoms with COVID-19 was not associated with viral RNA in the fecal
sample or extended duration of the viral RNA positivity in the feces.
COVID-19 RNA has been isolated from human saliva, nasopharynx and lower respiratory tract.
CT findings and lung abnormalities increased quickly after the onset of symptoms, and followed
by persistence of high levels in extent for a long duration. CT manifestations are important
inspected pattern over time [15]. A study was conducted that there is no evidence that TNF-α
inhibition will increase the risk of COVID-19 outbreaks, specifically [16].
Treatment:
At a moment, there is not yet any approved antiviral treatment for COVID-19. The
implementation of antiviral treatment and prophylaxis has several requirements to dip their risk.
Drugs can be administered shortly after symptom onset to reduce infectiousness and plummeting
viral shedding in the respiratory secretions. Some studied have approved hydroxychloroquine as
antiviral activity in vitro against coronaviruses, and specifically, COVID-19. Remarkably, this
drug was licensed for the chemoprophylaxis and treatment of malaria. Furthermore, drug testing
suggest that prophylaxis with hydroxychloroquine at approved doses may prevent COVID-19
infection and amend viral shedding [17]. Clinical trials of hydroxychloroquine treatment for
COVID-19 pneumonia have showed positive preliminary outcomes in China.
Unfortunately, corticosteroid treatment is commonly used in clinical practice for influenza virus
such as acyclovir, ganciclovir, ribavirin, and methylprednisolone as well as neuraminidase
inhibitors including (peramivir, oseltamivir, and zanamivir,) are invalid for COVID-19 and not
recommended [18].
Conclusions:
Currently, the scientists have made progress in characterizing the new coronavirus, and there are
still many questions that need to be answered. COVID-19 is the greatest biological hazard to
assume the relevance of insidious worldwide threat today. COVID-19 is a highly contagious
during the latency period. It is necessary may adopt and invest more modern technologies both to
facilitate notification, to allow speedier data dissemination and analysis in keeping with the
principles of precision epidemiology.
We suggest that close contact with an infected person is the major factor in disease transmission.
Healthcare workers also have to follow the CDC guidelines and should not attempt to perform
any virus isolation or characterization. The effect of mass gathering cancellations on reducing the
spread of COVID-19 needs to be determined. Current study conducted that any mutation
occurred in the former protein is especially important. There is no evidence that part of COVID-
19 is synthetic.
Declaration of competing interest:
The authors declare no conflicts of interest.

Acknowledgement:
At the end of this study, we would like to inform that all research budgets were self-supporting
without any institute donation.

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Another random document with
no related content on Scribd:
“Judges can be mighty nice, after all,” thought Charlie. “Still, I’d a
heap rather meet this particular one off the bench than on.”
“Now, boys,” spoke up Uncle Ralph, “a telegram must be sent to
your stout friend Brandon announcing our safe arrival. Tell Victor to
take a room at the hotel and expect me back to-morrow. Now, we’re
thirty odd miles from your motor car. Going with me in the morning,
or will——”
“Not for mine,” declared the “grind,” decidedly.
“Either Dave or Tom can drive the car,” said Bob. “So we’ll let ’em
come to us.”
“Very good.”
The hotel and restaurant which Uncle Ralph generally patronized on
his visits to the city was some distance from the wharf. As no
telegraph office was passed on the way they concluded to defer
sending the missive to Dave until after their meal. And this took
considerable time.
But the telegram was finally flashed over the wires with the request
that Dave should send an immediate response. Then nothing
remained but to see the sights and amuse themselves.
Captain Bunderley, after exacting a promise that they would meet
him at the hotel about six o’clock, returned to the yacht.
After they had wandered about the busy streets for some time
Charlie exclaimed:
“Now, what’s the program? My legs are beginning to put up a kick.”
“We are right close to that East Water Street bridge,” said Bob, as he
consulted a pocket map. “Looked like an interesting section to me.
Suppose we take it in?”
“One way is about as good as another, I s’pose,” replied Charlie,
wearily.
As the two came in sight of the bridge a tall, thin boy standing near a
little building at one end attracted Blake’s attention.
“By Jove!” he exclaimed, “I never expected to see another chap in
this part of the country with a shape just like Tom’s! What’s the
matter, Bob?”
Bob Somers’ expression had undergone such a sudden and startling
change that Charlie repeated his inquiry with a rising inflection.
“Don’t you recognize him?” demanded Bob, sharply.
“Recognize who?”
“Why, Tom—our Tom Clifton, of course!”
 CHAPTER XVI
TOM SCORES

“Oh, sugar! You’re dreaming. Pinch yourself,” cried Charlie Blake.

“Tom is miles from here; he’s away back in Kenosha, you silly
goose.”
“He was, but he isn’t; he’s right there in front of us.”
The “grind” gazed first at the tall boy, whose back was partly turned,
then toward his friend with such an air of comical bewilderment that
at any other time Bob Somers would have burst out laughing.
“It—it—certainly does look like Tom, but—but—why, hang it all, how
can it be Tom?” he gasped.
Bob Somers smiled, and the next instant Blake heard him utter a
lusty call which strangely resembled the hoot of an owl.
It produced a most extraordinary effect on the tall lad. He swung
around as sharply as though struck by some flying object. Then
Charlie heard an answering hail of a similar character, and, at the
same moment, saw the lad start toward them on a loping trot.
“Great Scott! It is—it actually is Tom Clifton!” he cried. “Well, well!”
On came the tall boy, while Bob Somers and his companion,
perhaps more astonished than they had ever been in all their lives,
walked rapidly to meet him.
Tom Clifton’s face, as he approached, presented a most curious
study. He made a desperate attempt to appear cool and dignified,
but, in spite of all his efforts, conflicting feelings of joy, triumph, and
even indignation persisted in finding reflection on his countenance.
“Well, Bob, I knew I’d see you!” was his exclamation, as he seized
the other’s hand. “That was a pretty slick scheme of Vic’s, but——”
“Slick scheme?” gasped Bob, while Charlie Blake’s mouth flew wide
open.
“Sure thing! Oh, you needn’t try to put on any nice innocent looks.”
Tom assumed an air of pitying condescension. “I got wise to your
dodge, all right; yes siree, Bob Somers. Ha, ha! You chaps didn’t get
up quite early enough to fool little Tom.”
“Why—why—what do you mean?” cried Bob. “How in thunder did
you get here, and why?”
“Well, that’s a good one!” exclaimed Tom, indignation suddenly
getting the upper hand of his other emotions. “Say, do you chaps see
anything green in me—ah, do you now?” A scornful look flashed in
his eyes. “Little Vic’s keeping out of sight, I suppose, eh? Thinks I
might hurt him. But—but—honest, Bob, I didn’t think it of you!” he
blurted out, unable to control his feelings any longer. “Honest, I
didn’t!”
“What does all this mean, Tom?” demanded Bob, sharply.
“Oh, now, cut it out, I tell you. I don’t mind a joke——”
“A joke?” broke in the highly mystified Blake.
“Yes; a joke! You understand English, I s’pose?”
“No; not this new brand of yours,” murmured Charlie.
“See here, Tom”—Bob Somers laid a hand on the other’s shoulder
—“let’s get at this thing. How did you come here?”
“In the motor car, of course.”
“And where are Dave and Victor?”
“Now look here, Bob,” cried Tom, hotly, “you and Charlie know—I
don’t. They helped you pull off this little trick and——”
“Great Cæsar! What kind of a mix-up is this?” cried Bob, a glimmer
of the true state of affairs entering his brain at last. “So you came
here alone?”
“A constable was in the car part way,” said Tom, loftily. “I let ’er out a
bit, Bob. And talk ’bout whizzing! Why, all the telegraph poles
seemed to be melted into one—honest fact, they did. Now tell me
what has become of Dave?”
“If Dave isn’t with you, he and Victor must be thirty-four miles from
here,” said Bob, calmly.
“What?” piped the tall lad, a sinking feeling suddenly gripping his
heart. Bob Somers’ expression was quite enough to convince him of
his sincerity. “Dave and Victor in Kenosha!” he added, faintly.
His thoughts ran riot for a moment. Then, after all, Victor Collins
wasn’t responsible. It really came as a stunning surprise to Tom.
“Well, Bob, the jinx has surely got us on this trip,” he exclaimed.
“Say, fellows, that was a foul tip of mine.”
Highly disgusted, Tom Clifton told the whole story, not forgetting,
even in his mental stress, to take credit for the fact that his
calculations regarding the destination of the motor yacht had proven
correct.
The “grind” was not demonstrative, as a rule, but on this occasion he
fairly roared with mirth, slapped his knees and grew so red in the
face that Tom became quite alarmed.
“Gee! Look out, Charlie,” he cautioned. “The system can stand only
so much.”
“I know; and this was just a trifle over the limit,” gurgled Blake. “Ha,
ha, Tom! You have Sherlock Holmes beaten a mile.”
Tom was highly aggrieved.
“I’ll leave it to Bob if anybody wouldn’t have been liable to think as I
did,” he declared, stoutly. “Now tell me how it happened that you’re
here.”
When Tom had been duly informed, Bob Somers remarked:
“Well, fellows, this certainly puts a new aspect on the case. What’s to
be done? Dave and Vic’ll think we’ve deserted ’em, sure. Another
thing: Dave didn’t have a cent.”
“Oh, that’s all right,” said Charlie. “Victor always carries nearly a ton
of the stuff in his pockets.”
“I’ll bet he wouldn’t lend Dave a nickel,” put in Tom, charitably.
“Looked to me as if he has it in for us.”
“Oh, get out,” scoffed Blake.
“Get off, you mean—eh, Bob?”
“We’ll most likely find a telegram waiting for us at the hotel,” said
Bob, shaking his head negatively at Tom’s suggestion.
“Let’s go and see.”
There was so much of interest in the streets that the boys didn’t feel
inclined to hurry themselves. So it was late in afternoon when they
finally set out in search of the hotel which Captain Bunderley
patronized.
By the time they reached it street lights and show windows were
gleaming brightly through the darkness of a very dark night.
The boys found Uncle Ralph in the reception room.
“Well, there’s no telegram from your friend yet,” was his greeting as
they stepped toward him. When his eyes lighted upon Tom Clifton’s
tall figure he half arose in his chair. “’Pon my word—what on earth
does this mean?” he exclaimed. “Where did you come from, boy?”
Tom was visibly embarrassed, as the eyes of every one in the room
were immediately leveled upon him.
“I blew in on the motor car,” he began, “and——”
“What—on the motor car—alone?”
“Yes, sir; and——”
“Let’s have this story right from the beginning,” thundered Uncle
Ralph, bringing his fist down on the table with a resounding bang, a
proceeding which added considerably to Tom’s confusion.
It wasn’t very easy for the tall boy to relate his story, especially with a
number of people sitting around, all apparently eager to hear him
speak. Uncle Ralph’s loud voice was the cause of this. He plunged in
bravely, however, being very careful indeed not to let out a hint
regarding Victor’s supposed trick.
Many and varied were Captain Bunderley’s observations as the tale
was told. The captain couldn’t help expressing his frank opinion at all
times, and in this case it wasn’t favorable to Tom.
“Why on earth did you do such a silly thing, boy?” he stormed.
“Silly?” cried Tom, aghast.
“Certainly; absolutely so.”
Tom, in helpless confusion, looked from Bob to Charlie.
“Silly?” he repeated, in fainter accents.
His face flushed a deep crimson. Then, suddenly, all the fire in his
nature flashed into a flame of burning indignation.
“It wasn’t a bit silly, sir,” he declared, fiercely.
“Now just see here, young chap”—the captain’s big finger waved
before Tom’s eyes; his voice boomed through the room with
appalling distinctness—“it was silly! What will Victor and Dave think
when they find you and the motor car missing?”
“I—I—don’t know, sir.”
“Of course you don’t. But just imagine how worried those two boys
may be.”
“Victor—perhaps; not Dave, sir. Besides, it isn’t my fault.”
“Not your fault?”
“No, sir. But for your running off with Bob and Charlie it never would
have happened.”
Tom came perilously near wilting under the captain’s stern gaze; only
by a desperate effort could he control his shaky nerves.
The lines on the skipper’s face softened; the harsh look faded from
his eyes.
“That’s true, my boy,” he said, reflectively; “quite true! Shake hands
and forget what I said. But the mischief must be undone at once.
Bob, I’m going to call up the hotel at Kenosha by long distance
’phone. My sister, if she knew the situation, I am sure would be
intensely worried about the boy.”
The three followed the captain’s burly form into the office.
Tom’s expression had undergone a most remarkable change; his
face now wore a look of conscious triumph.
“I squelched him some—eh, Bob?” he whispered in scarcely audible
tones. “He couldn’t make me the goat, oh, no!”
“Be with you in a moment,” bawled out the captain, entering a
telephone booth.
Little things like a closed door and a pile of boards couldn’t keep
Uncle Ralph’s voice within bounds. Presently they heard him say:
“What! Couldn’t give ’em the telegram because they’ve gone? How’s
that?—When? ’Pon my word! And left no message, either? Don’t
expect ’em back? Why not?”
The answer was evidently far from satisfactory, for, with a sharp “I’ll
call you up later,” Captain Bunderley flipped the receiver back into
place and stalked outside.
“Neither of ’em is at the hotel,” he exclaimed. “The clerk says they
went off at different times. Victor finally came back, but left again.
Says the stout boy asked for credit, but he was obliged to refuse.”
“Gee whiz!” cried Bob.
Then he promptly explained Dave’s situation, while Uncle Ralph’s
brow clouded over.
“A very annoying state of affairs, indeed,” he pronounced. “But let us
go in to supper, boys. Perhaps by the time we’re through some word
may have arrived.”
But it hadn’t. And when Uncle Ralph called up the Kenosha hotel a
second and third time the same laconic answer was always received
—“No, sir; they have not yet returned.”
“Well, that settles it,” cried Bob Somers, at length. “We’ll motor right
back to Kenosha and find ’em.”
“What!—On a night so black that a black cat would make a light spot
in the landscape?” exclaimed Captain Bunderley, protestingly.
“Oh, that kind of thing doesn’t worry us,” broke in Tom, eagerly.
“Why, when we were in Wyoming——”
“Oh, my!” groaned Charlie.
“Besides, it’s going to storm,” went on the captain, seeing a look in
Bob Somers’ eyes which indicated a settled determination.
“We have everything to protect us from the weather, sir. It’ll be a
regular lark. Coming, fellows?”
“Bet your life!” cried Tom, enthusiastically.
Charlie Blake, however, held back.
“Oh, look here, Bob, what’s the use?” he demurred. “We might miss
’em, and have all our trouble for nothing. It isn’t safe, either, traveling
——”
“Well, if you’re afraid, that ends it,” put in Tom, loftily.
“Who said I was afraid?” snapped the “grind.”
“Oh, nobody, of course,” said Tom, looking very wise.
“Then don’t chatter like a goose.”
“Did I ever hear of such nerve! If——”
“Cut it out, fellows,” laughed Bob. “Captain, we’ll send you a
telegram just as soon as those chaps are rounded up.”
The former seaman smiled quizzically. To his mind, talking about the
trip in a brightly-lighted room and actually undertaking it were two
such widely different propositions that he had little confidence in the
boys sticking to their determination. “They may possibly go as far as
the city limits,” he thought, “but that long stretch of lonely road and
the blackness will send ’em back.”
“Sure you want to try it?” he said, aloud.
“Yes, sir, the very worst way,” laughed Bob. “Now, Charlie——”

“LOOK OUT FOR YOURSELVES, BOYS”

Blake, anticipating what he was about to say, and noticing a peculiar
grin on Tom’s face, held up his hand.
“I’m going,” he said, in remarkably sour tones.
“That’s fine.”
Captain Bunderley gave each a hearty grasp of the hand.
“Look out for yourselves, boys,” he cautioned. “Remember: if I had
any authority over you I might not consent to your going.”
“Oh, don’t worry about us, sir,” said Bob. “We’ll be all right.”
“Down some embankment, I s’pose,” muttered Charlie.
Outside, Bob took a good look at the sky.
“I guess the skipper is right about the weather,” he remarked, as they
started off in the direction of the garage.
Half an hour afterward the three arrived at the building. It was a very
large garage containing many machines. The glare of electric lights
revealed none more imposing than the Rambler Club’s motor car.
“Not a speck o’ mud left on her,” said the man in charge. “Going out
to-night, sir?”
“Right away,” answered Tom, with an air of importance. “Pile in,
fellows.”
The fellows “piled” in.
“We may be back to-night, and we may not,” said the tall boy,
handing over the amount of the bill and a generous tip. “Let ’er go,
Bob. So-long!”
A deafening roar abruptly filled the whole room with thunderous
echoes. Quick gasps and throbs followed, while the exhausts flung
to the air whirling clouds of spent gases. The two head and two side
lamps threw a brilliant glare over the floor and walls and cut a
pathway through the open door to the street beyond.
The trembling machine, responding to its master’s touch, glided
forward.
“This sure ain’t no kind o’ a night for joy ridin’,” remarked the man in
charge, as he watched the big car swinging into the highway.
 CHAPTER XVII
ELEPHANTS

The boys didn’t turn back at the city limits as Captain Bunderley had
fully expected they would. Instead, the motor car finally passed
through South Milwaukee, and, under the cool and skilful guidance
of Bob Somers, plunged steadily along the muddy road, its lamps
throwing a strange, fantastic stream of light far in advance. Through
its magic, objects continually leaped out of the night, only to be
greedily snatched back by the mantle of gloom. Lights suggestive of
hobgoblins flitted from tree to tree, or swept across fields and
underbrush, but in the immensity of space beyond the glare
blackness held supreme control.
Heavy gusts of wind, moaning and whistling dismally in their hurried
flight, almost drowned the soft, even purring of the motor. Splashing
rain-drops hurled themselves against the wind shield and top; the
storm, long delayed, was beginning to let loose its pent-up wrath.
“Guess we’re going to have a peach of a time,” muttered Blake. “If I
hadn’t come, though, Tom would have kidded me about it for the
next six months.”
The disconsolate “grind” huddled back on the rear cushions listening
to the wind and rain and the soft swish of flying mud, as the rubber-
tired wheels occasionally plunged through pools and puddles.
“Let ’er out a bit, Bob,” encouraged Tom. “Don’t be afraid.” He pulled
the collar of his raincoat about his neck. “No constable around now
to stop our scorching. Gee! Ought to have seen me burning up the
road to-day, Bob; good you weren’t along, Charlie. Hey—asleep
back there?”
“How in thunder could a fellow be asleep with a din like this knocking
against his ear-drums?” growled Blake. “Where are we, Bob?”
“Somewhere between South Milwaukee and Racine—that’s all I
know,” answered the driver, with a laugh.
The wind blew harder; the rain, too, gradually increased in force until
sweeping torrents beat hard against the motor car, splashing its
occupants and forming tiny trickling pools in the bottom of the
tonneau.
Not a vehicle had passed them; the country seemed absolutely
deserted, and only dim points of light shining in the windows of
distant farmhouses indicated that any life existed in the seeming
wilderness.
The intense loneliness, the continual noises of the storm and the
haunting fear that hidden dangers might be lurking in their path
prevented Blake from entering into the spirit of the occasion.
“By Jove, this is certainly about the limit,” he groaned, inwardly.
From his position the forms of Bob Somers and Tom Clifton, bending
low to escape the cutting blasts, assumed a curiously unreal
appearance against the glare of acetylene light streaming ahead.
Leaning forward, he sought vainly to pierce the blackness; then, his
face becoming the target for splattering rain-drops, he hastily drew
back, to straighten up again a moment later as a shrill whistle sent a
series of wild reverberations across the landscape.
Over the air came faintly the rattle and roar of a fast express. The
road was taking them near the tracks of the Chicago and
Northwestern Railway. Charlie’s glance suddenly rested upon
something in the distance—a long row of tiny lights sweeping rapidly
toward them.
Now they disappeared; now flashed into view once more; the sound
of grinding car wheels rose higher. Then, with almost incredible
rapidity, the tiny lights became gleaming windows seeming to radiate
cheer as they sped onward through the night. In an instant more the
train was lost to view, and only a faint screech of the locomotive’s
whistle, fading quickly into the roar of wind and splash of rain, told of
its passing.
“Wish I was on board,” sighed Charlie. “Tom Clifton’s grins’ll never
drag me into any more silly adventures. This is ’most as bad as that
awful motor yacht trip. I’ve been going some to-day, all right.”
On the front seat, Tom was saying:
“Motoring in such blackness is dandy fun. The idea that you’re going
to run into something the next minute makes it kind of spicy, eh?
Gee, Bob, the rain’s coming down harder every minute. Wonder
where old Dave and Victor are now?”
“Very likely taking it easy in the hotel,” grinned Bob.
“Christopher! What’s that?”
A low rumbling sound had suddenly risen above the warring of wind
and rain.
“Thunder,” answered the chauffeur, briefly.
“Thunderation! I thought for a second it was a message from Dave
passing right over our heads,” laughed Tom. “Some weather, this,
Bob. Hello—a village beyond!—See it?”
Bob nodded.
“We’ll soon twirl that far behind us,” he said.
The faint points of light dotting the gloom gradually loomed up
stronger; the white glare from their lamps at length flashed over a
house by the roadside; then on another, and within a few minutes the
touring car was sweeping steadily through the village.
Out from the darkness a small form seemed to literally hurl itself
toward them, and, racing alongside, filled the air with vociferous
barks and yelps.
Leaning over, Bob saw a shaggy form of nondescript color, and
caught a gleam from a pair of greenish eyes.
“Nice doggie!” he chirped, soothingly.
“He’s started off every other ‘nice doggie’ in town,” chuckled Tom.
“Listen!”
The baying of numerous canines, some near at hand, others in the
distance, was rising on the air.
“Some up-to-the-minute constable may nab us for disturbing
sleeping dogs,” said Tom.
“That’s so,” grinned Bob.
He manipulated the lever. The car leaped forward, leaving their four-
footed foe far in the rear. For a few moments, his senses keenly alert
for any signs of danger, he kept up the swinging gait, slowing up as
the lights of a store and smithy close to it shot into view.
As they passed the latter a cheerful glow was spreading out over the
street from a partly-open door. The boys caught a momentary
glimpse of figures and horses within, and heard vigorous blows on
an anvil sending forth a series of musical notes.
Then the long street, silent and deserted, slipped slowly by, and,
presently, the motor car was threading its way in the zone where
human activities seemed to have ceased. Another stretch of dreary
blackness followed, with the trees, in the grip of the blasting air
currents, soughing and snapping their branches mournfully.
Pelting rain still assailed the travelers. The motor car often rolled
through deep pools, scattering sheets of muddy spray aside. The
boys could hear the oozy, sucking sound of slimy masses torn from
their resting places and spattering against the guards.
“Say, Bob, wouldn’t this be a great place for the machine to break
down?” came in a sepulchral voice from the rear.
“You’d surely lose the polish on your shoes, Charlie,” laughed Tom.
“Wonder what Vic ’ud say to this?”
“Oh, he’d let out an awful howl.”
“And no one could blame him, either,” growled the disgusted Blake.
On and on went the car, through another village and then another,
and, finally, the city of Racine was seen asserting itself strongly
against the gloom of nature.
The boys found on entering the town that most of the stores were
closed; but the brightly-lighted streets and the sight of electric cars
and an occasional pedestrian was a welcome change after their
siege of riding in the lonely country.
“Too bad we can’t stay here for a while, fellows!” exclaimed Bob, “but
it’s the long road and blackness again for us.”
“Dave and Vic are probably sound asleep by this time,” grumbled
Charlie, “never dreaming about the lovely time we’re having on their
account.” Then he added, softly, to himself: “Guess I’ll be having
nightmares about it, though, for weeks to come.”
“Speed her up, Bob,” said Tom, eagerly. “A chap can see where he’s
going out here.”
The street stretched straight ahead, with not a vehicle in sight. The
glare of electric lights flashed on steadily falling rain; the gutters ran
with miniature floods, which gurgled and splashed along, carrying on
their muddy surfaces a miscellaneous collection of rubbish. Here and
there great pools reflected the buildings and telegraph poles with
weird effect.
Bob put on more speed; the motor car leaped forward, and for
several blocks they flew ahead at a breath-taking pace. An electric
car coming from the opposite direction presently whizzed past, a
confused mass of blurred lights and shadows.
“This is simply great, Bob!” cried Tom. “Enjoying yourself, Charlie?”
he added, with a laugh.
The “grind” was not; so the only answer Tom’s query brought forth
was a dissenting grunt.
Occasionally Blake took a long, careful survey of the situation. On
looking out, a few moments later, he saw a residential section
passing before his eyes. This was quickly followed by the open
country and desolation.
The storm, which had lulled during a short period, broke forth with
renewed activity. At intervals coppery colored lightning streaked
across the heavens, or forked its way to earth. In the brief instants of
dazzling glare a series of singularly clear impressions, of dark,
twisting clouds, of distant farmhouses, of rail fences, of waving trees,
and of formless patches of shadow were imprinted upon Charlie’s
brain. It filled the boy with a curious sense of awe and dread which
refused to be shaken off.
“A bend in the road just ahead, Bob!” Tom at length sang out.
“I see it,” responded the chauffeur.
The advancing rays of light showed the broad road disappearing
around a mass of vegetation.
“Have to slow up now,” said Bob—“danger of the machine skidding
on slippery ground like this.”
“You bet.”
As the touring car slackened speed Bob sent forth a long, warning
blast of the horn.
Tom laughed.
“Gee, Bob, what was the sense of doing that?” he cried. “We haven’t
passed many drays and trucks, or——”
Blake, listening indifferently, would have continued to do so but for
the loud, startled exclamation which brought Tom’s sentence to an
abrupt close.
He looked up quickly, then, with a gasp of astonishment, he fell
forward, bringing up against the seat in front with a violent bump.
The motor car had scarcely swept around the bend when the
acetylene glare picked out from the darkness the forms of three huge
elephants advancing directly in their path. Almost stupefied with
amazement, the boys, at the same instant, saw two men walking
close beside them. Not far behind, the light shone upon a huge, red
wagon.
Before the warning cries which came from the men ceased Bob
Somers had thrown out the clutch and applied the brakes, bringing
the machine to a halt almost within its own length.
A shrill trumpet call sounded. The elephant in advance, showing
evidence of the greatest alarm, suddenly broke away from its keeper
and attempted to turn back.
Then followed a scene which made the nerves of the chauffeur and
his companions tingle with excitement. A huge pachyderm, wheeling
his body around, effectually blocked the other’s progress. Almost
immediately the third elephant got into action, while every movement
made by the men, in their efforts to pacify the animals, only added to
their fear and confusion.
“Great Scott!” breathed Charlie, grasping Bob by the arm.
The three towering forms were swinging wildly toward them, the
nearest threatening to plunge full tilt against the motor car.
 CHAPTER XVIII
A ROUGH TRIP

“Victor, wouldn’t it be better for you to skip back to the hotel?”

asked Dave, looking anxiously at the sky.
The lawyer’s son thought of the dark, gloomy streets through which
he would be obliged to pass; then the idea of actually traveling with a
circus appealed strongly to his imagination.
“No, Brownie,” he answered, decidedly.
“Joe,” said Dave, turning toward the circus boy, “I see the light of a
drug store over yonder; guess they have a ’phone. I’m going to call
up the hotel. Can you wait, Joe?”
“Sure, Dave. But if Whiffin ketches me busy at doin’ nothin’ it means
a callin’ down—see?”
“All right, Joe; we’ll hurry,” said Dave, encouragingly.
“An’ while you’re gone I’ll help git the elephants ready,” announced
Joe, with sudden decision. “Them three old codgers goes ahead o’
us.”
Dave, followed by Victor, loped across the wet, soggy lot, or, rather,
tried to. But, although the journey was attended by much discomfort
and some risk of taking a header, they finally arrived at the drug
store in safety.
Dave promptly called up the hotel and was soon speaking to the
night clerk. The latter declined to open the telegram, but gave the
stout boy full information about the ’phone message which Captain
Bunderley had sent from Milwaukee.
“Well?” queried Victor, eagerly, as the historian hung up the receiver.
Dave briefly explained.
“There, you big Indian, I knew it!” exclaimed the lawyer’s son,
triumphantly. “A nice trick they played on us, eh? Well, I’m liable to
handle that Tom Clifton with awful carelessness when we meet
again. Now, Brownie”—his tone became imperious—“you just call up
Uncle Ralph on the long distance and tell him what’s what.”
With a broad smile, the stout boy obeyed.
To his disappointment, however, he was told that Captain Bunderley
had retired for the night.
“If it’s important we’ll get him right up for you,” came a faint voice
over the wire.
Dave did some rapid thinking. “Poor Joe is most likely fretting and
fuming about the delay,” he mused. “Besides, if I wait any longer
there may be another mix-up.”
He spoke in the transmitter again:
“Thank you; I’m in too much of a hurry. Will you kindly take down a
message and give it to the captain at once.”
The distant clerk assured him that he would. Dave quickly went over
the few facts which he thought it was necessary for the captain to
know, ending with: “He’ll hear from me in the morning.” “Good-bye”
was trembling on his tongue when an afterthought prompted him to
ask: “How many boys are in the party?”
“Three,” came the answer.
“One very tall?”
“Yes, sir.”
“Any of them about?”
“No; all went out together some time ago.”
“Thank you. Good-bye.”
“What did he say?” demanded Victor.
“Yes; Tom is there, all right.” Dave smiled. “Come on,” he added,
seeing the familiar expression of anger instantly flash into the other’s