The Division Algorithm

- ₁₃₄

-CBAPTBR 5

- 13�

WN virus strains by both neutra1•-ation .u. aDd CF tests, rosults ot routine HI tests showed general broad

reactions 11mong the ceven strainc. Bowovor, varying the reaction time of the antigen n.nd antibody as in

Casals moditiod HI toat, tho usual pattern of rolation

shipo noticed in other tests woo oeen, ocpocially in the two hour reaction time. This n& an indication that roactions botween BA antigon and ito antibody were more specific ^at shorter reaction time than ^at longer reaction time when the reactions become

generally broad.

The reeulto of ^agar-gel precipitation testo clearly showed that totnl identity ^existed botwoon otrains 4029 and 30046. Tho reoulte also revealed closo relationohip aoong ctrains 7019, 23001, 41402, 41504 and 50247. The tqo�way roactione were quite prominent. In offoct, tho existence of two antigenic fP'OUpa baeod on cross reactions ^with strains 4029 and 7019 rospectively bad boon firot revealed by •Bar-gol precipitation teats.

Growth cbaracteriatioo of ^'ITif viruo strains in

mice will appear to ^{be a} difficult index for separating the strains into different antigenic groupo. Generally, infection in mice is ⁸ ystoMio and tho infoctivity titres

.. , /130

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130 -obtained tor each strain appeared to be of the same level in oaoh orean. Strains 4029 ^and 30045 bo,rever, because of the time of appearance ot peak iotectivity titres in the brain, especially and in other organo will appear to suggest tho close relationships between the two strains. Odelola and O'Coooor (1972) showed

that with antigens produced in mouse ascitic fluid peak infoctivity titres for strain 4029 appeared at tho aamo timo as for strains 0029 and 38045 in these studies. These observed differences could not be attributable to adaptation since oacb strain used in these studies bas bad same passaae history in mice.

Plaque production by five related strains 7019, 23001, 41402, 41504 and 502�7 ao againot non

production of plaques by two identical strains 4029 and 30015 showed that ple.que production could be uaod to characterise virus strains. The ability of tho five related strainc to produco plaquec and tho in

ability of tho two identical strains to develop plaques agroed with the pattorn of antigenic rolationohips

already noticed in other tosts. However, it is known that ability to develop plaquoo in tioauo culture is a genetic property of infective viral particl es .

(Dulbecco, 1952). Plaque development ao revoalod in ... /137

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137 tbe result obtained in tbe�e � studiee could, thoreloro, be associated with antigenic differencea. This io an indication of genetic differences _between the two

antigenic groups.

Elution of the ooven strainu of ^'ffN viru� ^r�

calciun phosphate column as o menne ot further chn,·1.c

terisation revealed that tbe cethod may not be �

suitable one to detect difteroncoo o.mong viruo strains.

Tho patterns of elution appeared generally identical with hiaheat elution ot viruo taking place at tho third traction 0.2Y in each of tho coven otraina.

Several workero had, howover, applied this method to ohow difference• among eertain viruoos. Smith and Bolt (op. cit.) usod the tl8thod to differentiate between groups A and B arbovirusos. Koza (1963)

employed tho method to identity virulent and avirulont strains of polio virus, while Ozaki ot al (1905)

applied tbe method tor the identification of vaccine progeny strains of polio viruo. The results obtaiood by thio cethod made it impoosiblo to differentiate the seven ^t1N v1ruc otrnins into two antiaonic groupo as have been done in other teats.

Throughout thees studios II.IIA.F trom mice given _two t •-us wore uoed ao source ot antibody.

injections o v ...

. .. /130

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'

- 130

-The ⁱ dea behind using such I!IAF was to take adY&ntaae of the fact that early ant ⁱ bodies which aro rich 1n itlmunoglobulin ^M (IaM) are usually core specif ⁱ c in the ⁱ r reactions than hyperimrune fluids which hl\ve broader react ⁱ ons. It io appP.ront, therefore, that

the use of two-injection I!l!APs had allowed the differen

tiation of the Higorinn strains of WN v.lruc into two serolog ⁱ cal ⁱ ntratypic groups depeodont on thoir cr?ss react ⁱ ons ^with stra ⁱ no 4029 ^and 7019 respectivoly.

Obviously all the seven strains shared a COQJi\On ant ⁱ gen, honco thoy could bo identifiod as stra ⁱ n.a of ^lffl virus

i n the first instance. In addition, aoc.o ot the strains possess a second antigen which related tbom 0oro closely to one or the other of tho prototype otraioo. 'l'his

coooopt is supported by the findines of Si.rlth and Uolt (op. cit.) that groups A and B arboviruseo have two baemagglutin ⁱ na wh ⁱ le group B arboviruses possess two complement fixing ant ⁱ gens. West Nilo virus is ^a group

B arbovirus and the antigon ⁱ o variation noticed amona the Nigerian strains confirmed the findingo of those workers. From tho broad reaotiono obtained in rospect of BI toots one is tompted to nasumo that the two

bllomaeglutinino poasessed by tboco stra ⁱ ns of YIN virus could bo r.ntisenically si. ^mi l&r · The naturo of tho

... /139

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139 -baecagBlutininE bad not been investigated but it ia 111,ely that one of the two haelilagglutinins could be the viruc particle. Tbo results obtained in both tho CF and neutralization testc also revealed tbAt two antigens were involved in eaeb case. Although the

nature of these antigens had not been well investigated, it is possible that one of them is a oomntio antigen

while the other ono is a aolublo one. Probably coCllllon West Nile antigen as bas been shown for Influenza

virus (Fabiyi et al 1950) is the soluble antigen.

The influence that tho sito of isolation (geographical location), source of infection and time of the year when isolation of the Nigerian

strains of WN virus woro cado need to be considorod in the light of observed antigeoio variation among these strains. Strains 4029 I\Dd 30045 have shown

unique relationships different from the other atraino under study. Thoso two virus strainc were isolated from sentinel mice at tho beginning of rainy season in Ibadan (Southern Nigeria) in a rain forest zone which falls between latitudes 6 and O north. Straioc 7019 and 23601 were also inolated in the uame locality ao the above two strains but at the peak of tho rainy season in tho months of August and July and from black

.•. /140

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110 -drongo bird and sentinel mouse respectively. These two latter strains ho , wever, showed strong serological relationships with strains 41402, 41504 and 50217

which were also isolatod at tho poak of raiDy acnson in Kano and ^Kware (Northern Nieeria) bet1:1oon ^!:. t L �•.1doa 11 and 13 north from Camels (41402 r.nd 4;_50,;) •�na

grass mouse (Arvicanthie nilotiuuo) roG�co�!voly.

Kano is a highly populated city in Nor�ho>·n Nigeria; it is an important centre for congrea�tion of traders who travel across tho Sahara dosert from Sudan, Egypt and other oountrien. It io interesting to state that strains 41402, 41504 and 50247 isolated from Northern Nigeria and strains 7019 and 23001 from Southern Nigeria have all shown close serological

relationships and have been confirmed to cross react with the Egyptian strain of WN viruo. One can only speculate that these strains wore imported into

Nigeria probably via infected camels or their ticks.

(Chumacov et al isolated WN virus from Ryalomn plumbeum.) These could sorve ao rosorvoirs for further trDllsmission to mosquitoes, birds and other animnls, especially

cattlo. The lattor could then, on migrating to Southorn Nigeria, sorvo as reservoir trom which mosquitoes and

•.. /141

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141 -other vectors can traruamt the

tible boats in the area. virus to otber auecep

Th is hypothesis could certainly explain the presence of strains 70l.9 and 23601 &nd thoir relationship to throe strains of IN virus isolated in Northern Nigeria.

Strains 4029 and 3C045 uniquely have consistently shown non-relationship to thoso strains isolated from Northern Nigeria and which in turn a.re related to the Ecyptian strain, These two unique strains can,

therefore, be considered to bo indigenous to Southern Niaoria, It does not nppoar that the season plays any major role on the lllltigenic differenceo observed in the WN virus strains under study, ainoo ^all of tho virus strains woro isolated during the rainy season.

The only role that source of isolation could ploy in these studies is that of poasiblo ^animAl migration, especially tho infected ones which could introduce

new virus strains into the country.

Hawnam et al (op.cit.) have classified ^lfN isolates into threo zoogoographic zones: tho Palo.ea.retie,

Oriental and Ethiopian &roups. The palnearctic strains include European and Hiddle-Eaat ioolntoa, tho oriental is made up of Indian isolates, while the Ethiopian

group includes isolates from areas south of tho Sahua.

, .. /142

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- 14.2

-According to the above grouping, Nigerian ^isolates should ^fall into the Ethiopian gToup. The ^results,

however, show that five related isolates of tho ^Nigeria.a strains of WN virus i:.re ant1.genically rolt.ted 1.c- the

Egyptio.o isolates whic�ll bolo:iit to the general p11lo.oai-c

tic gTOup. Two etraina, 4029 nod 300<15, fo.11 to oh ^., , any antit;onic roln.tioll'ibi\l to vlrUD str".11! frr>:tJ 1·ho tbroo zoogeogTo.phic ^arPab. ·rhese f1ndiU!l3 cent ⁱ ro tho observations of RyAn n:1d Casals (Veraonal CoCl!llW)1o�t1on) that strain 4029 is Wlique and crooe !:'eact� leoG ^•1 th any of tho strains from tho three zoogoog:ro.phic ^nreae and that strains 7019 and 23601 fall into the r,onoral palaearctic gTOUP,

The existonco of antigenic variation a�ong strains of WN virus in Nicerie. is of orncticcl uiportuco. The choice of a strain to be uAed �s � &crooning antigen for the detection of aorum 111nr.it:cdy must be cl\l'oful.l;: con

sidorod in serologico.l su>:veys. it 7ou1�1 appo•r that a strain isola tod frOJ!I �be e.ro• Ju 11·h ic:b tho sora woro collectod would serw, l>oct, but tbJs :i.a r.ot al"'!YfJ

feasible and pro.oticnl. Theso stuJies had coofirood the above since one ^of tbo strnios 1solctod in Southern Niseria bad been ablo to pick up more sera positivo to YN virus in Northern and southern Nigeria. Tho practical

... /143

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- 143

-importance ot these studies was tb&t it had been

possible to choose a suitable strain (7019) which can be uaed as a screening antigen for the detection of serUD antibody to WH virus in future serological

surveys in Nigeria. llacnam.ra et al (op. cit.) in a sorological survey of sora collocted in South-Western Nigoria found that the parcentago of sera positive to WM virus antibody na very low. This could be due to

the use ot an unsuitable strain as a &crooning antigen.

Tho uso of strain 7019, therefore, ^{as a} screenins ^anti sen could load to more accurate results being recorded in such serological surveys.

Finally, it bas to be stressed that tho purpose of initiating these studies as bad boon done by other workers like Karabatsos ot al (1963), Clarke (1962) and Casals (op. cit.) to mention just a fow, was to determine whother antigenic ovidonoe would strengthen or weaken the concept of rapid spread of a virus over

a wide geogro.pbic area through animal and bird migration.

Sorologic differences between strains of a givon virus aro of importance in the idontificatioo of newly isolated arboviruses. Inadequate knowledge of antigenic

differon-i i en ^Vir us could load to erronous cha.rac-ces with n a g v

... /14,

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- 144

-characterization of an agent as a new virus and not merely a strain of a recogniced v1ruo.

. •. /145

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145 -Cllf,n&t 0

Strd!IARY AllD CONCLUSIONS

Ucing sero-jrarnunological techniques, eight isolates ot Nigerian strains ot ^\TN viruo ^were investieated to

determine antieonic rolationshipo among them and to find out ^it tbeco viruoeo were related to WN virus ctrains froa the different zooceoerapbio areas of the world. One virus, str!lin 20COC., differed si8n1ficaJJtly

from the sevon other ctrains and was lntor concidered to be Uautu viruo rather than ^ilN viruo.

Detniled anclysoc of tbe recaining seven etraino of virus isolntoo revenled two ooroloaicnl intratypic L'Toupc. Tbis D'oupina ,ro.o bacod on croso-roactiono vith 2 other strains �bicb served no prototypas for

each sub--group. 3tra1nc •1020 and ^3C045 which ^wero oricinnlly isolated froc the rain foroot zone of

NieoriA comprised a cub-group uniquely serologically related but quite dictinct froc tho cocond sub-group which tnnludes s-trninc 7019, 23001, �lGO?., 11504 and 50247. The latter sub-group wao found to bo related

... /146

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146 -to the strain ot Wll virus origi.nating from general palaen.rctic zoogeoe:rapbic zone (European and W.ddle Bast). Theoretical considerations of tbe origin

and tbe spread of virus strain.a fr011 tbe general palaearctio zone were explored and it was suggested that tbeae straiDtJ must have been b;ougbt to Nigeria from d01118stic animals such as caJ11Gls and further

opread by birds and ouoceptible arthropod vectors.

It was found tbnt for epidemiologic studios, ocrooning of sora nod soro-<iiagnoois, strain 7019 wbich had a broad spectrum of reactivity with tho

other otrains wns a most suitablo strain for detecting nnd evnluoting antibodies to WH virus. With this. ^1fN strain,sera wbicb were found to ^be nog11tive to otber strains were contirm�d to have antibodies to WN virus.

The new antigenic sub-group, strains 4029 and

30045, which did not belong to either the Palaoarctic, Oriental or Ethiopian group appeared uniquo so far to Nigoria only and pooeiblY to Wost Africa in general.

Further cbaractorisotion and tho proporties of these strains ohould prove to ho of groat interost and

- 134

-CBAPTBR 5

- 13�

WN virus strains by both neutra1•-ation .u. aDd CF tests, rosults ot routine HI tests showed general broad

reactions 11mong the ceven strainc. Bowovor, varying the reaction time of the antigen n.nd antibody as in

Casals moditiod HI toat, tho usual pattern of rolation­

shipo noticed in other tests woo oeen, ocpocially in the two hour reaction time. This n& an indication that roactions botween BA antigon and ito antibody were more specific at shorter reaction time than at longer reaction time when the reactions become

generally broad.

Growth cbaracteriatioo of 'ITif viruo strains in

mice will appear to be a difficult index for separating the strains into different antigenic groupo. Generally, infection in mice is 8 ystoMio and tho infoctivity titres

.. , /130

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130

Plaque production by five related strains 7019, 23001, 41402, 41504 and 502�7 ao againot non­

production of plaques by two identical strains 4029 and 30015 showed that ple.que production could be uaod to characterise virus strains. The ability of tho five related strainc to produco plaquec and tho in­

ability of tho two identical strains to develop plaques agroed with the pattorn of antigenic rolationohips

already noticed in other tosts. However, it is known that ability to develop plaquoo in tioauo culture is a genetic property of infective viral particl es .

(Dulbecco, 1952). Plaque development ao revoalod in ... /137

UNIVERSITY OF

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137

tbe result obtained in tbe�e � studiee could, thoreloro, be associated with antigenic differencea. This io an indication of genetic differences between the two

antigenic groups.

Elution of the ooven strainu of 'ffN viru� r�

calciun phosphate column as o menne ot further chn,·1.c­

terisation revealed that tbe cethod may not be �

suitable one to detect difteroncoo o.mong viruo strains.

Tho patterns of elution appeared generally identical with hiaheat elution ot viruo taking place at tho third traction 0.2Y in each of tho coven otraina.

Several workero had, howover, applied this method to ohow difference• among eertain viruoos. Smith and Bolt (op. cit.) usod the tl8thod to differentiate between groups A and B arbovirusos. Koza (1963)

employed tho method to identity virulent and avirulont strains of polio virus, while Ozaki ot al (1905)

applied tbe method tor the identification of vaccine progeny strains of polio viruo. The results obtaiood by thio cethod made it impoosiblo to differentiate the seven t1N v1ruc otrnins into two antiaonic groupo as have been done in other teats.

Throughout thees studios II.IIA.F trom mice given two t •-us wore uoed ao source ot antibody.

injections o v ...

. .. /130

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'

- 130

-The i dea behind using such I!IAF was to take adY&ntaae of the fact that early ant i bodies which aro rich 1n itlmunoglobulin M (IaM) are usually core specif i c in the i r reactions than hyperimrune fluids which hl\ve broader react i ons. It io appP.ront, therefore, that

the use of two-injection I!l!APs had allowed the differen­

tiation of the Higorinn strains of WN v.lruc into two serolog i cal i ntratypic groups depeodont on thoir cr?ss react i ons with stra i no 4029 and 7019 respectivoly.

Obviously all the seven strains shared a COQJi\On ant i gen, honco thoy could bo identifiod as stra i n.a of lffl virus

i n the first instance. In addition, aoc.o ot the strains possess a second antigen which related tbom 0oro closely to one or the other of tho prototype otraioo. 'l'his

coooopt is supported by the findines of Si.rlth and Uolt (op. cit.) that groups A and B arboviruseo have two baemagglutin i na wh i le group B arboviruses possess two complement fixing ant i gens. West Nilo virus is a group

B arbovirus and the antigon i o variation noticed amona the Nigerian strains confirmed the findingo of those workers. From tho broad reaotiono obtained in rospect of BI toots one is tompted to nasumo that the two

bllomaeglutinino poasessed by tboco stra i ns of YIN virus could bo r.ntisenically si. mi l&r · The naturo of tho

... /139

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IBADAN LIBRARY

139

-baecagBlutininE bad not been investigated but it ia 111,ely that one of the two haelilagglutinins could be the viruc particle. Tbo results obtained in both tho CF and neutralization testc also revealed tbAt two antigens were involved in eaeb case. Although the

nature of these antigens had not been well investigated, it is possible that one of them is a oomntio antigen

while the other ono is a aolublo one. Probably coCllllon West Nile antigen as bas been shown for Influenza

virus (Fabiyi et al 1950) is the soluble antigen.

The influence that tho sito of isolation (geographical location), source of infection and time of the year when isolation of the Nigerian

strains of WN virus woro cado need to be considorod in the light of observed antigeoio variation among these strains. Strains 4029 I\Dd 30045 have shown

.•. /140

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110

-drongo bird and sentinel mouse respectively. These two latter strains ho , wever, showed strong serological relationships with strains 41402, 41504 and 50217

which were also isolatod at tho poak of raiDy acnson in Kano and Kware (Northern Nieeria) bet1:1oon !:. t L �•.1doa 11 and 13 north from Camels (41402 r.nd 4;_50,;) •�na

grass mouse (Arvicanthie nilotiuuo) roG�co�!voly.

relationships and have been confirmed to cross react with the Egyptian strain of WN viruo. One can only speculate that these strains wore imported into

Nigeria probably via infected camels or their ticks.

(Chumacov et al isolated WN virus from Ryalomn plumbeum.) These could sorve ao rosorvoirs for further trDllsmission to mosquitoes, birds and other animnls, especially

cattlo. The lattor could then, on migrating to Southorn Nigeria, sorvo as reservoir trom which mosquitoes and

•.. /141

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IBADAN LIBRARY

141

-other vectors can traruamt the

tible boats in the area. virus to otber auecep­

Th is hypothesis could certainly explain the presence of strains 70l.9 and 23601 &nd thoir relationship to throe strains of IN virus isolated in Northern Nigeria.

Strains 4029 and 3C045 uniquely have consistently shown non-relationship to thoso strains isolated from Northern Nigeria and which in turn a.re related to the Ecyptian strain, These two unique strains can,

therefore, be considered to bo indigenous to Southern Niaoria, It does not nppoar that the season plays any major role on the lllltigenic differenceo observed in the WN virus strains under study, ainoo all of tho virus strains woro isolated during the rainy season.

The only role that source of isolation could ploy in these studies is that of poasiblo animAl migration, especially tho infected ones which could introduce

new virus strains into the country.

- ₁₃₄

Casals moditiod HI toat, tho usual pattern of rolation

shipo noticed in other tests woo oeen, ocpocially in the two hour reaction time. This n& an indication that roactions botween BA antigon and ito antibody were more specific ^at shorter reaction time than ^at longer reaction time when the reactions become

Growth cbaracteriatioo of ^'ITif viruo strains in

mice will appear to ^{be a} difficult index for separating the strains into different antigenic groupo. Generally, infection in mice is ⁸ ystoMio and tho infoctivity titres

Plaque production by five related strains 7019, 23001, 41402, 41504 and 502�7 ao againot non

production of plaques by two identical strains 4029 and 30015 showed that ple.que production could be uaod to characterise virus strains. The ability of tho five related strainc to produco plaquec and tho in

tbe result obtained in tbe�e � studiee could, thoreloro, be associated with antigenic differencea. This io an indication of genetic differences _between the two

Elution of the ooven strainu of ^'ffN viru� ^r�

calciun phosphate column as o menne ot further chn,·1.c

applied tbe method tor the identification of vaccine progeny strains of polio viruo. The results obtaiood by thio cethod made it impoosiblo to differentiate the seven ^t1N v1ruc otrnins into two antiaonic groupo as have been done in other teats.

Throughout thees studios II.IIA.F trom mice given _two t •-us wore uoed ao source ot antibody.

-The ⁱ dea behind using such I!IAF was to take adY&ntaae of the fact that early ant ⁱ bodies which aro rich 1n itlmunoglobulin ^M (IaM) are usually core specif ⁱ c in the ⁱ r reactions than hyperimrune fluids which hl\ve broader react ⁱ ons. It io appP.ront, therefore, that

the use of two-injection I!l!APs had allowed the differen

tiation of the Higorinn strains of WN v.lruc into two serolog ⁱ cal ⁱ ntratypic groups depeodont on thoir cr?ss react ⁱ ons ^with stra ⁱ no 4029 ^and 7019 respectivoly.

Obviously all the seven strains shared a COQJi\On ant ⁱ gen, honco thoy could bo identifiod as stra ⁱ n.a of ^lffl virus

coooopt is supported by the findines of Si.rlth and Uolt (op. cit.) that groups A and B arboviruseo have two baemagglutin ⁱ na wh ⁱ le group B arboviruses possess two complement fixing ant ⁱ gens. West Nilo virus is ^a group

B arbovirus and the antigon ⁱ o variation noticed amona the Nigerian strains confirmed the findingo of those workers. From tho broad reaotiono obtained in rospect of BI toots one is tompted to nasumo that the two

bllomaeglutinino poasessed by tboco stra ⁱ ns of YIN virus could bo r.ntisenically si. ^mi l&r · The naturo of tho

which were also isolatod at tho poak of raiDy acnson in Kano and ^Kware (Northern Nieeria) bet1:1oon ^!:. t L �•.1doa 11 and 13 north from Camels (41402 r.nd 4;_50,;) •�na

tible boats in the area. virus to otber auecep

therefore, be considered to bo indigenous to Southern Niaoria, It does not nppoar that the season plays any major role on the lllltigenic differenceo observed in the WN virus strains under study, ainoo ^all of tho virus strains woro isolated during the rainy season.

The only role that source of isolation could ploy in these studies is that of poasiblo ^animAl migration, especially tho infected ones which could introduce

Hawnam et al (op.cit.) have classified ^lfN isolates into threo zoogoographic zones: tho Palo.ea.retie,

-According to the above grouping, Nigerian ^isolates should ^fall into the Ethiopian gToup. The ^results,

however, show that five related isolates of tho ^Nigeria.a strains of WN virus i:.re ant1.genically rolt.ted 1.c- the

Egyptio.o isolates whic�ll bolo:iit to the general p11lo.oai-c

The existonco of antigenic variation a�ong strains of WN virus in Nicerie. is of orncticcl uiportuco. The choice of a strain to be uAed �s � &crooning antigen for the detection of aorum 111nr.it:cdy must be cl\l'oful.l;: con

feasible and pro.oticnl. Theso stuJies had coofirood the above since one ^of tbo strnios 1solctod in Southern Niseria bad been ablo to pick up more sera positivo to YN virus in Northern and southern Nigeria. Tho practical

Tho uso of strain 7019, therefore, ^{as a} screenins ^anti sen could load to more accurate results being recorded in such serological surveys.

differon-i i en ^Vir us could load to erronous cha.rac-ces with n a g v

Ucing sero-jrarnunological techniques, eight isolates ot Nigerian strains ot ^\TN viruo ^were investieated to

determine antieonic rolationshipo among them and to find out ^it tbeco viruoeo were related to WN virus ctrains froa the different zooceoerapbio areas of the world. One virus, str!lin 20COC., differed si8n1ficaJJtly

from the sevon other ctrains and was lntor concidered to be Uautu viruo rather than ^ilN viruo.

each sub--group. 3tra1nc •1020 and ^3C045 which ^wero oricinnlly isolated froc the rain foroot zone of

other otrains wns a most suitablo strain for detecting nnd evnluoting antibodies to WH virus. With this. ^1fN strain,sera wbicb were found to ^be nog11tive to otber strains were contirm�d to have antibodies to WN virus.